Amrit Kaur Bansal

Cooperative functions of manganese and thiol redox system against oxidative stress in human spermatozoa.

AIMS: In this study, the effects of 0.1 mM Mn2+ on thiol components (total thiols [TSH], glutathione reduced [GSH], glutathione oxidized [GSSG] and redox ratio [GSH/ GSSG]) have been determined in human spermatozoa. SETTINGS AND DESIGN: The subjects of the study were healthy males having more than 75% motility and 80 × 106 sperms/mL. MATERIALS AND METHODS: Fresh semen was suspended in phosphate-buffered saline (PBS) (pH 7.2) and this suspension was divided into eight equal fractions. All fractions, control (containing PBS) and experimental (treated/untreated with [ferrous ascorbate, FeAA - 200 FeSO4 μM, 1000 μM ascorbic acid, nicotine (0.5 mM) and FeAA + nicotine], supplemented/unsupplemented with Mn2+ [0.1 mM]), were incubated for 2 h at 37°C. These fractions were assessed for determining the thiol components. STATISTICAL ANALYSIS: The data were statistically analyzed by Students “t” test. RESULTS AND CONCLUSIONS: Ferrous ascorbate, nicotine and ferrous ascorbate + nicotine induced oxidative stress and decreased GSH and redox ratio (GSH/GSSG ratio) but increased the TSH and GSSG levels. Mn2+ supplementation improved TSH, GSH and redox ratio (GSH/GSSG) but decreased the GSSG level under normal and oxidative stress conditions. Thiol groups serve as defense mechanisms of sperm cells to fight against oxidative stress induced by stress inducers such as ferrous ascorbate, nicotine and their combination (ferrous ascorbate + nicotine). In addition, Mn2+ supplementation maintains the thiol level by reducing oxidative stress.

Seminal Plasma Heparin Binding Proteins Improve Semen Quality by Reducing Oxidative Stress during Cryopreservation of Cattle Bull Semen.

Heparin binding proteins (HBPs) are produced by accessory glands. These are secreted into the seminal fluid, bind to the spermatozoa at the time of ejaculation, favour capacitation, acrosome reaction, and alter the immune system response toward the sperm. The present study was conducted with an objective to assess the effect of purified seminal plasma-HBPs (SP-HBPs) on cross bred cattle bull sperm attributes during two phases of cryopreservation: Pre freezing and freezing-thawing. SP-HBPs were purified from pooled seminal plasma by heparin affinity chromatography. Three doses of SP-HBPs i.e. 10, 20, 40 μg/mL semen were standardized to find out the optimum dose and 20 μg/mL was found to be an optimum dose. Semen as such and treated with SP-HBPs was diluted with sodium citrate-egg yolk diluter and cryopreserved as per the standard protocol. Sperm parameters i.e. motility, viability, Hypo-osmotic swelling test (HOST), acrosome damage, in vitro capacitation and lipid peroxidation were evaluated in SP-HBP treated and untreated (control) semen at both phases of cryopreservation. A considerable variation in percent sperm motility, viability, membrane integrity (HOST), acrosome damage, acrosome reaction and lipid peroxidation was observed at both phases among the bulls irrespective of the treatment. Incubation of neat semen with 20 μg/mL SP-HBP before processing for cryopreservation enhanced the average motility, viability, membrane integrity by 7.2%, 1.5%, 7.9%, and 5.6%, 6.6%, 7.4% in pre-frozen and frozen-thawed semen in comparison to control. There was also an average increase of 4.1%/3.9% in in vitro capacitation and acrosome reaction in SP-HBPs-treated frozen-thawed semen as compared to control. However, binding of SP-HBPs to the sperm declined acrosome damage and lipid peroxidation by 1.3%/4.1% and 22.1/32.7 μM/109 spermatozoa in SP-HBP treated pre-frozen/frozen-thawed semen as compared to control, respectively. Significant (p<0.05) effects were observed only in motility, HOST and in vitro acrosome reaction. It can be concluded that treatment of neat semen with SP-HBPs before cryopreservation minimized the cryoinjury by decreasing the generation of reactive oxygen species.

Characterization and immunolocalization of HBP, FA-1 and TIMP-2 like proteins in cattle bull semen: HBP modification in in vitro capacitated spermatozoa

Characterization and localization of HBP, FA-1 and TIMP-2 like proteins in spermatozoa and seminal plasma of cattle bulls was carried out by immunoblotting and immunofluoresence. Anti-HBP, anti-TIMP-2 and anti-FA-1 reacted with 55, 48, 45, 42, 35, 30, 24, 18, 16 kDa; 65, 45, 24, 16 kDa and 55, 48, 16 kDa sperm proteins on immunoblots. Immunofluoresence indicated that HBP/TIMP-2 are localized mainly on acrosomal cap, whereas, FA-1 predominantly on post acrosomal cap. Among the bulls, positive for 60, 45, 16 kDa FA-1 like proteins in sperm extracts and 11/16 kDa in SP; 7, 8, 6 and 7 bulls also showed higher rate of in vitro AR. Number of bulls positive for 65, 24 kDa-TIMP-2 and with higher rate of AR was more as compared to other anti-TIMP-2 reactive sperm/SP proteins. Therefore, 60, 45 and 16 kDA-FA-1, 65 and 24 kDa-TIMP-2 like proteins may serve as indicators of higher rate of in vitro AR vis a vis fertility of cattle bulls. Immunoblotting of cpapacitatd and un-capacitated spermatozoa with anti-HBP suggested that removal of 55, 48, 45, 40, 37 and 30 kDa HBP from in vitro acrosome reacted cattle bull spermatozoa allowed heparin to mediate in vitro AR and an increase in intensity of 110, 90 kDa and exposure of 65, 60, 26 and 16 kDa HBP after AR may be important for binding of sperm to ovum, penetration and fertilization.

Antioxidants and Other Potent Strategies to Reduce Oxidative Stress in Semen

Oxidative stress (OS) has been considered a major contributory factor to the male infertility. It is the result of imbalance between the reactive oxygen species (ROS) and antioxidants in the body which can lead to sperm damage, deformity, and eventually male infertility. Although high concentrations of the ROS cause sperm pathology (ATP depletion) leading to insufficient axonemal phosphorylation, lipid peroxidation, and loss of motility and viability, but many evidences demonstrate that low and controlled concentrations of these ROS play an important role in sperm physiological processes such as capacitation, acrosome reaction, and signaling processes to ensure fertilization. ROS are also generated during cryopreservation of spermatozoa for AI practices. To reduce the oxidative stress, there are certain compounds and reactions which dispose, scavenge, and suppress the formation of ROS or oppose their actions are called antioxidants. Currently, many antioxidants are under investigation. The supplementation of a cryopreservation extender with antioxidant has been shown to provide a cryoprotective effect on mammalian sperm quality. This chapter explains the impacts of oxidative stress and reactive oxygen species on spermatozoa functions, causes of ROS generation, and antioxidative strategies to reduce this OS. This study also suggests that antioxidant supplementation could be of clinical importance in prolonging the spermatozoal storage for assisted reproductive techniques (ARTs) like artificial insemination (AI), in vitro fertilization (IVF), and intrauterine insemination (IUI) purposes.

Purification and Characterization of Heparin Binding Proteins from Seminal Plasma of Cross- bred Cattle Bulls by Affinity Chromatography, SDS-PAGE and Mass Spectrometry

Heparin binding proteins (HBP) play a crucial role in the fertility of bovine semen. In this study HBPs purified from cross-bred cattle bull seminal plasma (SP) by sepharose-affinity chromatography were characterized by SDS-PAGE, and mass- spetrometery. Affinity chromatographic analysis indicated two peaks of unbound (non-HBP) and bound proteins (HBP). On average, seminal plasma of cross-bred bulls contained 39.36 ± 4.41% HBP with a peak area of 2.74 ± 0.82 cm2. Sixteen bands with molecular weights ranging from 14 kDa to 150 kDa could be separated by SDS-PAGE from seminal plasma of 11 bulls. SDS-PAGE analysis of the eluted HBP peaks identified 14 bands, with molecular weights ranging from 14 kDa to 150 kDa. However, variation in number of bands, separated in SP and SP-HBP was observed among the bulls. The matched peptides of 60; 40, 35; 31, 28; 25 and 20 kDa proteins with highest score (>61-67) were identified to have significant matching with the peptides of Platelet activatin factor AH; Clusterin preprotein; DNase1-L3 and TIMP-2, respectively. This study envisaged that four characterized SP-BHPs have important functions in reproduction. Moreover, role of DNASE-1L-3 and TIMP-2 kDa proteins is related to higher conception rate of bovine. Therefore, this study opens a further scope to analyze these SP – HBP as potential biomarkers of fertility in cross – bred bulls.

Mutual interaction of dog sperm LDHC4, PH-20, actin and tubulin proteins and their immunocontraceptive potential in bitches

In this paper, interaction of LDHC4, PH-20, a-actin and b-tubulin on dog’s sperm surface and their immunocontraceptive effect in bitches has been elucidate. Anti spam -1 and anti LDHC4 recognized 46/32 and 36/30/28 kDa proteins in dog sperm extracts on immunoblots. Whereas, proteins of 43, 36 kDa and 62, 46, 43, 30 and 28 kDa were detected on immunoblots with anti-a-actin and anti-b-tubulin. In MALDI-TOF analysis, sequence of 46, 30, 28 and 43, 36 kDa antigens matched twice to b-tubulin and a-actin respectively. Therefore, one sub unit each of LDHC4 and a-actin had same mol wt of 36 kDa and two other sub units each of LDHC4 and b-tubulin shared the same mol wt of 30/28 kDa. Reaction of 46 kDa protein both with anti-spam-1 and b-tubulin indicated similarity in their mol wts. Similarly the reaction of 43 kDa protein both with anti-a-actin and anti- b-tubulin revealed some similarity between both. Sequence analysis revealed 19.49%, 24.28%, 21.42% and 18.75% similarity of LDHC4/ a-actin, PH-20/ a-actin, LDHC4/ b-tubulin and LDHC4/tubulin respectively. Immunofluoresence staining of dog sperm smears also indicated similarity in localization of these four proteins. Localization of PH-20 and b-tubulin was mainly on the entire head region. Whereas, LDHC4 is uniformly distributed on the whole sperm and actin is more concentrated on the post acrosomal cap. Purified native proteins of 46/32 and 36/30/28 kDa were injected to a group of two bitches respectively through i/m route to elucidate their immunocontraceptive potential. Immunization of bitches in both the groups resulted in suppressed heat, and inhibited natural mating. On being subjected to AI, ultrasonography of four immunized and two unimmunized (control) bitches 35 days after insemination confirmed the presence of 0 and 3-4 fetuses respectively. Therefore, it seems that LDHC4/PH-20 and a-actin/ tubulin share their molecular weights or interact with each other or actin/ b-tubulin superimpose LDHC4 /PH-20 on sperm surface. In view of observed contraceptive effect, it can be concluded that LDHC4 and PH-20 collectively with a-actin and b-tubulin affect estrus, process of natural mating and ultimately the fertility in bitches.