Short-term outcome of magnesium sulfate infusion in perinatal asphyxia.

This randomized, single blind, controlled, clinical trial was done to see the effect of magnesium sulfate infusion in perinatal asphyxia. This study was conducted in the Department of Neonatology, Bangabandhu Sheikh Mujib Medical University and Dhaka Medical College Hospital from January, 2010 to October, 2010. Total 50 term neonates having postnatal age less than 12 hours with history of perinatal asphyxia and had history of hypoxic ischemic encephalopathy (moderate or severe) were included in this study. Patients were assigned randomly to receive either 3 doses of magnesium sulfate infusion at 250mg/kg per dose (0.5ml/kg per dose) 24 hours apart (experimental group) or 3 doses of normal saline infusion 24 hours apart (placebo-controlled group). Both groups also received supportive care according to the unit protocol for perinatal asphyxia. Baseline characteristics of 50 neonates had no differences in gestational age, birth weight, gender, mode and place of delivery, parity, ANC, liquor colour and hypoxic-ischemic encephalopathy (HIE) staging and mean age of intervention between the experimental and controlled groups. The mean serum magnesium at admission was 1.6±0.3mg/dl and 1.8±0.4mg/dl and after 48 hours was 3.9±0.6mg/dl and 1.9±0.2mg/dl in experimental group and in controlled group respectively. There was no significant difference or alteration in colour, heart rate, respiration, capillary filling time/blood pressure and oxygen saturation between the experimental and control groups. At discharge, 26% (5 of 19) of infants in the experimental group had neurological abnormalities, compared with 61% (11 of 18) of infants in the control group. At discharge experimental group were received more (78% vs. 44%) oral feedings by sucking compared with the controlled group. There is no significant difference in Electroencephalographic (EEG) abnormalities between groups. Good short-term outcomes at discharge were seen more (60% vs. 32%) in the experimental group, compared with the placebo-controlled group. The overall mortality rate in our study was 26%. Postnatal magnesium sulfate infusion is effective in improving short-term outcomes in neonate with perinatal asphyxia.

Pott's disease in a young child.

A 16-month-old emaciated, immunized child presented with low-grade fever and progressive kyphosis with a right sided para-vertebral abscess for 9 months. During this period of illness the child had marked loss of appetite and progressive weight loss. There was history of contact with sputum smear positive father. A gibbus was present at thoraco-lumbar region with a cold abscess at the right side of the gibbus and signs of upper motor neuron lesion were found on lower limb examination. Diagnosis was supported by relevant investigations including MRI of dorsolumbar spine and treatment was started beforehand with anti-tubercular drugs. The paravertebral abscess was drained several times and antibiotics were used depending upon the results of microbiological study. At the same time the patient was advised to wear a modified chest brace for immobilization and the management for severe malnutrition was also started accordingly. There was significant clinical improvement observed within one month of starting treatment.

Multiple glutathione S-transferase isoforms are present on male germ cell plasma membrane.

Phase II detoxification enzymes, the glutathione S-transferases (GSTs) of 24 kDa are known to be cytosolic enzymes. This study shows that multiple GST isoforms that are 24 kDa in size are present on the extracellular side of the plasma membrane of rat male germ cells. The GST activity of male germ cell plasma membranes is several folds higher than somatic cell plasma membrane GST activity. Isoform composition of the germ cell plasma membrane and the cytosolic pool differ, GSTM5 and GSTPi being absent on the plasma membranes. The molecular masses of the common isoforms are comparable between the two pools and both pools show GST and glutathione peroxidase activity.

Hydrogen peroxide induces apoptosis-like death in Leishmania donovani promastigotes.

Leishmania donovani promastigotes introduced into the bloodstream by sandfly vectors, are exposed to reactive oxygen species like H2O2 during phagocytosis by the host macrophages. H2O2 can induce promastigote death, but the mechanism of induction of this death is not known. Studies presented in this paper demonstrate that exposure to 4 mM H2O2 results in a pattern of promastigote death that shares many features with metazoan apoptosis. Motility and cell survival in these parasites show a gradual decline with increasing doses of H2O2. Features common to metazoan apoptosis, such as nuclear condensation, DNA fragmentation with accompanying DNA ladder formation and loss of cell volume, are observed after exposure to 4 mM H2O2. Within 30 minutes of the exposure, there is a significant increase in the ability of the cell lysates to cleave the fluorogenic tetrapeptide acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin, which is a substrate for the CED-3/CPP32 group of proteases. Pretreatment of cells with a specific inhibitor of CED-3/CPP32 group of proteases, Z-DEVD-FMK, reduces the number of cells showing apoptosis-like features, prevents DNA breakage and inhibits cleavage of a PARP-like protein. Activation of the caspase-like proteases is followed at 2 hours by the cleavage of a poly(ADP)ribose-polymerase-like protein and a reduction in intracellular glutathione concentration. DNA breakdown as detected by TdT labelling of cells and agarose gel electrophoresis is visible at 6 hours. Taken together, the above data show for the first time that there is a distinct pathway for apoptosis-like death in L. donovani.

Role of glutathione S-transferases in oxidative stress-induced male germ cell apoptosis.

Cellular apoptosis in a tissue may occur for the maintenance of proper ratio of cells or because of toxic effects of free radicals or other agents. Male germ cell apoptosis is pivotal in maintaining the proper functioning of the testis, but it is not clear how free radicals affect germ cells and what the defense mechanisms are that are used by these cells to combat the toxic effects of the products of oxidative stress. This study shows that male germ cells are susceptible to H(2)O(2)-induced stress and, upon exposure to H(2)O(2) in vitro, demonstrate a typical apoptotic phenotype that includes DNA fragmentation and formation of DNA ladders. Other changes include considerable accumulation of products of lipid peroxidation in the germ cells after exposure to H(2)O(2). Evidence is presented for the existence of multiple isoforms of glutathione S-transferases (GSTs) that possess both transferase and Se-independent peroxidase activity. Germ cell GST activity increases after H(2)O(2) exposure. If this increase in activity is inhibited with suitable inhibitors, the formation of products of lipid peroxidation is augmented, resulting in germ cell apoptosis. Also, when constitutive GST activity is inhibited, accumulation of products of lipid peroxidation occurs, resulting in increased cellular apoptosis. These data show that GSTs form a part of adaptive response of germ cells to oxidative stress and are important constituents in detoxifying the products of lipid peroxidation.

Experimental unilateral undescended testis: gubernaculectomy and anchoring or direct suture fixation?

This study was designed to evaluate whether creation of a unilateral undescended testis (U/L UDT) in rats by direct fixation of the testis can lead to changes in the contralateral (C/L) descended testis, and if so, whether this inherent problem of the model could be eliminated by anchoring the divided gubernaculum to indirectly fix the testis. Thirty male newborn rats were divided into three groups of 10 each and the procedure done on the 2nd day of life to create U/L UDT according to the group allocated: group I: sham-operated; group II: anchoring the gubernaculum after gubernaculectomy; group III: Direct suture fixation of the testis. Fertility, C/L testicular weight (TW), Johnsen score, seminiferous tubular diameter (STD), DNA flow cytometry, and serum anti-sperm antibodies (ASA) were studied. Fertility, C/L TW, Johnsen score, STD, and haploid cell population were significantly reduced in group III compared to group II, while significantly higher titers of ASA were found in group III. Gubernaculectomy and anchoring the gubernaculum to the anterior abdominal wall is a better technique of creation of experimental UDT, as direct fixation of the testis is potentially detrimental to the C/L normal, descended testis.

Secretion of glutathione S-transferase isoforms in the seminiferous tubular fluid, tissue distribution and sex steroid binding by rat GSTM1.

The seminiferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis in the adluminal compartment of the seminiferous tubule (ST), primarily through secretions of the Sertoli cell. Earlier studies from this laboratory demonstrated the presence of glutathione S-transferase (GST) in STF collected from adult rat testis and in the spent media of ST cultures. This study describes the cellular source, isoform composition and possible function of GSTs in the STF. The major GST isoforms present in STF in vivo share extensive N-terminal similarity with rat GSTM1 (rGSTM1), rGSTM2, rGSTM3 and rGST-Alpha. Molecular masses of rGSTM2, rGSTM3 and rGST-Alpha from liver and testis sources were similar, unlike STF-GSTM1, which was larger by 325 Da than its liver counterpart. Peptide digest analysis profiles on reverse-phase HPLC between liver and STF isoforms were identical, and N-terminal sequences of selected peptides obtained by digestion of the various isoforms were closely similar. The above results confirmed close structural similarity between liver and STF-GST isoforms. Active synthesis and secretion of GSTs by the STs were evident from recovery of radiolabelled GST from the spent media of ST cultures. Analysis of secreted GST isoforms showed that GST-Alpha was not secreted by the STs in vitro, whereas there was an induction of GST-Pi secretion. Detection of immunostainable GST-Mu in Sertoli cells in vitro and during different stages of the seminiferous epithelium in vivo, coupled with the recovery of radiolabelled GST from Sertoli cell-culture media, provided evidence for Sertoli cells as secretors of GST. In addition, STF of 'Sertoli cell only' animals showed no change in the profile of GST isoform secretion, thereby confirming Sertoli cells as prime GST secretors. Non-recovery of [35S]methionine-labelled GSTs from germ cell culture supernatants, but their presence in germ cell lysates, confirm the ability of the germ cells to synthesize, but not to release, GSTs. Functionally, STF-GSTM1 appeared to serve as a steroid-binding protein by its ability to bind to testosterone and oestradiol, two important hormones in the ST that are essential for spermatogenesis, with binding constants of <9.8x10(-7) M for testosterone and 9x10(-6) M for oestradiol respectively.

Fertility and unilateral undescended testis in the rat model II.

Continuing experimental work on the effect of experimentally created unilateral undescended testis (UL-UDT) in neonatal rats, this study examined the fertility and correlated it to contralateral (CL) testicular morphology, seminiferous tubular diameter (STD), DNA flowcytometry, and the presence of serum anti-sperm antibodies (ASA) at 120-135 days of age. In our previous reported work, the fertility of rats with UL-UDT at 65-80 days of age was the same as that of controls. In the present study the rats with UL-UDT had significantly reduced fertility (P < 0.01) compared to controls, even though the Johnsen scores and mean STD of the CL testicular tissue were comparable. DNA flowcytometry demonstrated a significant decrease (P < 0.001) in haploid cell population in the CL testicular tissue of rats with UL-UDT. Furthermore, the rats with UL-UDT who either received an immunosuppressive or in whom the UDT was excised early showed almost normal fertility and DNA histograms like those of controls. Significantly high titres of serum ASA were detected only in the group with UL-UDT when tested at 135 days of age. From these results, in combination with earlier results on similar work, it may be inferred that UL-UDT causes immunologically-mediated, progressive damage to the CL descended testis, leading to a decrease in fertility in rats.

Inhibition of sperm glutathione S-transferase leads to functional impairment due to membrane damage.

The role of glutathione S-transferase (GST) in the defense mechanisms of sperm is not known. We report here interference with normal motility, acrosome reaction and fertilizing ability of the goat sperm as a consequence of inhibition of GST activity. That these functional impairments were due to membrane changes was evident from the alteration in the lipid peroxidation status of these cells after GST inhibitor treatment. Increased reactive oxygen species production by the cell which occurred when GST activity was suppressed may be the mediator for membrane damage. The data argue for a role of GST in maintaining sperm membrane status.

Studies on glutathione S-transferases important for sperm function: evidence of catalytic activity-independent functions.

Our earlier studies reported the identification of a rat testicular protein of 24 kDa with significant similarity at the N-terminus with Mu class glutathione S-transferases (GSTs). Treatment of goat sperm with antisera against this protein identified immunoreactive sites on the spermatozoa and inhibited in vitro fertilization of goat oocytes by the antibody-treated sperm. The above observations indicated the presence of GST-like molecule(s) important for fertility related events on goat spermatozoa. In this study, we report the purification of goat sperm GSTs (GSP1) which were purified by glutathione affinity chromatography and were enzymically active towards 1-chloro-2,4,-dinitrobenzene, a general GST substrate, and ethacrynic acid, a substrate for Pi class GSTs. GSP1 resolved into three major components on reverse-phase HPLC: peaks 1 and 2 with molecular masses of 26.5 kDa and peak 3 with a molecular mass of 25.5 kDa, as determined by SDS/PAGE. Multiple attempts to obtain N-terminal sequences of the first two peaks failed, indicating N-terminal block; however, they reacted to specific anti-Mu-GST antisera on Western blots and ELISA, and not to anti-Pi-GST antisera, which provides evidence for the presence of Mu-GST-reactive sites on peaks 1 and 2. The third component showed 80% N-terminal similarity with human and rat GSTP1-1 over an overlap of 15 amino acids, and reacted to anti-Pi-specific antisera in ELISA. Sperm labelled with antibodies against a 10-mer and an 11-mer peptide, designed from the N-terminal sequences of Mu and Pi class GSTs respectively, showed the presence of both Mu- and Pi-GST on goat sperm surface at distinct cellular domains. Selective inhibition of Pi class GST by the Pi-specific antisera, either at 0 h or at 3 h after initiation of sperm capacitation, leads to a reduction in fertilization rates. In contrast, the inhibition of Mu class GST by specific antisera at 0 h does not inhibit fertilization, although such treatment at 3 h after the initiation of capacitation reduces fertilization rates. The results indicate that both Pi- and Mu-GSTs are involved in fertilization, but the Mu-GST sites essential for fertilization are exposed only after 3 h of capacitation. The enzymic activity of GSP1 or live spermatozoa is not inhibited by the two antisera. The inability of the antibodies to cause such inhibition indicates that the reduction in fertilization rates and acrosome reaction caused by the antibodies is through a mechanism which does not interfere with the catalytic activity of the molecule. Therefore we established the presence of Pi and Mu class GST on goat sperm, their localization and their possible function in fertility-related events.

A testicular protein important for fertility has glutathione S-transferase activity and is localized extracellularly in the seminiferous tubules.

A 24-kDa protein isolated by preparative gel electrophoresis from rat testes was reported by us as an active immunogen in rats. Anti-24-kDa antibodies inhibited murine sperm-oocyte binding in vitro. Here, we show similarity at the NH2 terminus shared by this protein purified on Sephadex G-75 followed by anion exchange high performance liquid chromatography with glutathione S-transferase (GST)-mu subunits. This protein purified by glutathione affinity chromatography also demonstrated similarity to GST-mu NH2 terminus in a 30-amino-acid overlap. Both proteins showed activity toward the GST substrate 1-chloro-2,4-dinitrobenzene (Km of 33 microM and 50 microM) which was inhibited by 17 beta-estradiol 3-sulfate. Antisera against both proteins recognized liver GST-mu on Western blots and sperm acrosome of multiple species immunocytochemically. Both antisera significantly inhibited in vitro fertilization of goat oocytes by sperm preincubated with them while anti-liver GST sera did not. GST activity was localized on rat sperm, seminiferous tubular fluid, and Sertoli cells. Seminiferous tubular fluid 24-kDa protein shared similarity to the NH2 terminus of GST-mu subunits in a 20-amino-acid overlap. Time-dependent accumulation of GST was detected in the spent culture medium of seminiferous tubules from rats of different ages suggesting secretion.

Antibody-induced changes on rabbit sperm surface inhibit gamete interaction.

Interaction of specific ligands with cell surface molecules may induce reorganization of surface components. A monoclonal antibody (B-12) against sperm surface antigens of 40kDa size induced molecules on the plasma membrane overlying the acrosome of rabbit sperm to cluster in small aggregates at 0 degree C (patching). At an elevated temperature of 37 degrees C these clusters of antigen antibody complexes collected into a large aggregate on one pole of the cell forming a cap (capping). This cap-like structure showed a reduction in size over a period of time and eventually disappeared from the sperm surface. Inhibition of capping by sodium azide indicated that it is an energy-dependent process. Patching of antigens did not require energy. Involvement of sperm head cytoskeleton in the process of capping was evident from potentiation of cap formation by cytoskeleton disrupting agents like cytochalasin B and D. Patching of antigen antibody complexes was not affected by either of the agents. The loss of antigen antibody complexes from sperm surface was mainly due to shedding of the complexes in the surrounding media. Sperm with patches of antigen antibody complexes did not adhere to oocytes. Sperm from the group where a majority of the sperm were denuded of the antigen antibody complexes also did not bind to oocytes.

Monoclonal antibody against a human sperm protein recognizes multiple epitopes on rabbit and human sperm and blocks sperm function.

A monoclonal antibody was raised against a human sperm protein of apparent molecular size of 40 kDa. Of the 6 hybridoma clones selected for study, one clone (B-12) was chosen for further investigations. The antibody secreted by the clone agglutinated human and rabbit spermatozoa in vitro. The antibody belonged to IgM class. In indirect immunofluorescence studies this antibody reacted to acrosome of living human and rabbit spermatozoa. On fixed sperm of the same species it recognized midpiece and parts of tail along with the acrosome. Mouse, hamster, guinea pig, monkey and rat sperm showed similar localization. Interaction between rabbit sperm and oocyte was inhibited in vitro by this antibody. On western blots of human and rabbit sperm extracts the antibody recognized more than one epitope.

Amphiphilic and hydrophilic domains of human sperm membrane antigens recognized by immunoinfertile sera.

Selected sera from married couples with immunological infertility were used to identify antigens on hydrophilic and amphiphilic domains of human sperm membrane. Out of eight sera, six recognized proteins from the hydrophilic as well as amphiphilic regions of the sperm membrane. Sera were either reactive to acrosome or to equator and tail of human sperms in indirect immunofluorescence assay.

Vaccines for control of fertility.

PIP: Major developments in birth control vaccines are on the horizon. The human chorionic gonadotropin (hCG) vaccine has entered phase II clinical trials after successful completion of phase I studies at 5 centers in India and 4 centers abroad. It is the most advanced vaccine of its type in the world. The trials are being conducted on women of proven fertility who are sexually active. The available results indicate the efficiency of the vaccine to prevent pregnancy in women at or above titres of 50 ng/ml. A vaccine inducing antibodies against gonadotropin releasing hormone (GnRH) has been approved in India for trials in postpartum women, to determine whether immunization can help prolong lactational amenorrhea. The GnRH vaccine is also in clinical trial in prostate cancer patients at 2 centers in India and in Austria and the Dominican Republic. The follicle stimulating hormone (FSH) vaccine is about to enter phase I clinical trial after completing experimental and toxicological studies. A vaccine against FSH has been developed for human males employing ovine FSH (oFSH) as an immunogen. oFSH adsorbed on alum induces antibodies reactive with human FSH in bonnet monkeys. Immunization leads to oligospermia with resultant impairment of fertilization potential. No reduction in testosterone levels has been reported. Research is in progress to identify antigens on spermatozoa, which could serve as vaccine candidates. PH-20, a protein located on the inner acrosomal membrane of capacitated sperms, has been reported to have 100% contraceptive efficacy in both sexes of guinea pigs in active immunization studies. cDNA probes of PH-20 cross-react with genomic DNAs of mouse, rat, hamster, and human. The sperm antigen, lactate dehydrogenase C4 (LDH-C4), is a glycolytic enzyme. Active immunization with LDH-C4 suppressed fertility in mice, rabbits, and baboons. SP-10, which is a testis-specific human sperm protein, is also a promising candidate.^ieng

Characterisation of 24-kD proteins from rat testes using polyclonal sera reactive to human sperm antigens.

A group of antigens of 24-kD Mr from rat testes were characterised biochemically. These antigens were part of a larger molecule of approximately 200 kD. On treatment with disulfide bond reducing agent, the 200-kD molecule was reduced to subunits. Immunoreactivity was confined to a doublet of approximately 24 kD and a single band of approximately 50 kD Mr after the reduction. Glycoprotein in nature, this antigen shared immunoreactive epitopes with a 40-kD antigen on human spermatozoa. Antiserum raised in rabbits against the 24-kD antigen from rat testes reacted with antigens on the acrosome of human spermatozoa. Agglutination of sperm could be induced by the antiserum. The carbohydrate residue could be removed by mannosidase digestion. Chemical deglycosylation studies showed a slight decrease in molecular weight. Immunoreactivity was however not completely lost after chemical deglycosylation. Isoelectric focusing of the antigen identified nine isoelectric species. Two relatively minor species showed immunoreactivity. Acrosome-reacted spermatozoa showed loss of antigens from acrosome.

Induction of infertility in female rats after active immunization with 24 kD antigens from rat testes.

A unique polyvalent antiserum against whole washed human sperm was used previously to identify groups of antigens on spermatozoa. The antiserum, designated as Antiserum I, recognized a 40 kD antigen in human sperm extracts. Antiserum I caused agglutination of human sperm and prevented interaction of mouse sperm and oocytes. This serum also recognized a band of 24 kD in rat testicular cytosol. In the present study this group of 24 kD proteins was used as an antigen preparation to actively immunize female rats. Immunization was carried out with two different adjuvants: nor-muramyl dipeptide and SPLPS (a thyalated derivative of lipopolysaccharide). Both groups of animals showed significant antibody titres as detected by indirect immunofluorescence and by sperm agglutination tests. In both groups over 80% of the animals remained infertile, compared to 13% of the controls. It is concluded that a group of antigens in rat testes recognized by Antiserum I offer promise as candidates for a contraceptive vaccine.

Characterization of a monoclonal antibody against a 24KD antigen from rat testis important for fertility regulation.

Monoclonal antibodies were raised against a 24KD antigen from rat testicular cytosol which was previously shown to produce antibody titres in rats with inhibitory effect on fertility. Of the 20 hybridoma clones selected for study, one clone HS-D5 was finally chosen for characterisation as it proved to be functionally promising. The clone was secreting IgM type of antibody. It produced strong agglutination of human sperm and prevented binding of hamster sperm to hamster oocyte. In localization studies it identified the acrosome of sperm of multiple species. Western blots with rat testicular cytosol and human sperm extracts showed a strong band at around 24KD with HS-D5. On Western blots from two dimensional gels, HS-D5 identified multiple spots at the region of 24KD. The mechanism of action of the antibody seemed to be at the level of interaction of the oocyte and spermatozoa.

Immunostainable inhibin subunits are in multiple types of testicular cells.

Immunostainable inhibin alpha-subunit has been demonstrated in rat testes in a pattern consistent with localization in Sertoli cells. In the present study the distribution of alpha-subunit immunostaining was compared to those of beta-A- and beta-B-subunits. Immunostaining of alpha-subunit was present in the seminiferous epithelium of fetal, neonatal, pubertal, and adult rats as well as in Sertoli cells in culture. The distribution of inhibin beta-B-subunit immunostaining in this epithelium was consistent with Sertoli cell localization similar to that of the alpha-subunit. The predominant staining with antibodies against the beta-A-subunit was in nuclei of immature germ cells around the periphery of each seminiferous tubule. The most probable localization of this staining was in the nuclei of pachytene and zygotene spermatocytes. Specific immunostaining with beta-A-subunit antiserum was also evident in the seminiferous epithelium adjacent to the tubular lumen. Immunoreactive alpha- and beta-A-subunit staining was present in a Leydig cell line, and beta-A immunoreactivity was present in interstitial cells of neonatal rat testes. After hypophysectomy, inhibin alpha-subunit immunostaining decreased, beta-A-subunit staining did not change, and beta-B-subunit staining increased. We conclude that immunoreactive inhibin subunits are present in multiple cells in the testis and that the amounts of immunostainable subunits in the seminiferous epithelium are differentially regulated.