Intracellular delivery of peptide cargos using polyhydroxybutyrate based biodegradable nanoparticles: Studies on antitumor efficacy of BCL-2 converting peptide, NuBCP-9.

Faster biodegradation, immunogenicity and lack of cell penetrative capabilities are hurdles in development of peptidyl drugs for cancer therapy. Polymeric carriers can be used to overcome these problems. The present study is focused on the use of polyhydroxybutyrate as a potential nanovehicle for the delivery of anticancer peptides. PHB (72kDa) was produced by thermal treatment of high molecular weight PHB (300kDa) under melt conditions and then conjugated with PEG (4kDa) by Steglich esterification reaction. Anticancer peptide NuBCP-9 (FSRSLHSLL) encapsulated PHB(72K)-PEG(4K) NPs were prepared by double emulsion solvent evaporation method. PHB(72K)-PEG(4K) NPs showed encapsulation efficiency of 61% and exhibited sustained release of peptide over a period of 26days at physiological pH. NuBCP-9 loaded PHB(72K)-PEG(4K) NPs showed an IC50 value of 2.2µM & 1.6µM in MCF-7 cells in 48h and 72h respectively. Confocal laser microscopy confirmed efficient cellular uptake and induction of apoptosis by peptide loaded NPs in a time dependent manner. In vivo intraperitonial administration of 20mg/kg NuBCP-9/NPs twice a week for three weeks triggered 90% tumor regression in Ehrlich syngeneic mouse model. Our results illustrated the potential of PHB(72K)-PEG(4K) based nanoformulation as a tool for targeting intracellular proteins.

Protective effects of Eugenia jambolana extract versus N-acetyl cysteine against cisplatin-induced damage in rat testis.

To assess the protective effects of Eugenia jambolana extract (EJE) or N-acetyl cysteine (NAC) on testis, cisplatin (CIS, 5 mg kg(-1) bw, single dose) was administered either alone or along with EJE (25 mg kg(-1) bw, alternate day) or NAC (150 mg kg(-1) bw, Day 1 and 4) for 7 days. Significant alterations in serum LH, FSH and testosterone were observed in CIS group which were effectively modulated by EJE or NAC supplementation. Upregulation of 3ß-HSD gene indicated the rise in functional Leydig cells. This was further confirmed from the identical improvement in hCG-stimulated testosterone production in isolated Leydig cells. Reduction in oxidative stress was associated with restoration of total antioxidant capacity and glutathione levels, and activation of antioxidant enzymes, SOD, catalase, glutathione s-transferase (GST) and glutathione reductase (GR). CIS-induced apoptosis of germ and Leydig cells was contained by both NAC and EJE intervention by effective modulation of apoptotic markers in the extrinsic, intrinsic and other pathways of metazoan apoptosis. Taken together, the study findings establish the potential of EJE as a therapeutically better antioxidant than NAC for use in curtailing the adverse effects of anticancer drugs on testicular function.

Quercetin supplementation restores testicular function and augments germ cell survival in the estrogenized rats.

Quercetin, as a flavonoid, has been recognized to possess dual properties of an oxidant and antioxidant as well. The role of quercetin (QC), as an antioxidant in countering estradiol-3-benzoate (EB) induced adverse effects and germ cell apoptosis in adult rat testis was presently investigated. Adult rats received EB (0.075 mg/rat/5th day) alone or EB+QC (15 mg/kg bw/alternate day) simultaneously for 30 days. Revival of spermatogenesis following QC intervention was associated with a significant restoration in serum and intra-testicular levels of testosterone. Decline in lipid peroxidation and simultaneous improvement in the activities of superoxide dismutase, catalase and glutathione s-transferase were very much evident. Identically, total antioxidant capacity and glutathione demonstrated a marked improvement. QC augmented germ cell survival leading to a decrease in cell apoptosis. Expression of downstream apoptotic markers, caspase-3 and poly-ADP-ribose polymerase (PARP) presented a significant reduction. Down regulation with respect to upstream markers, caspase-8 and -9, Fas, FasL, Bax, and p53 was similarly observed. Taken together, the above findings indicate that with the dose presently used quercetin with its antioxidant and antiestrogenic properties restored testicular function leading to revival of spermatogenesis. It also augmented germ cell survival primarily mediated through downregulation in the expressions of upstream, downstream and other markers in the pathways of metazoan apoptosis.

Clomiphene citrate potentiates the adverse effects of estrogen on rat testis and down-regulates the expression of steroidogenic enzyme genes.

OBJECTIVE: To investigate the antiestrogenic effect of clomiphene citrate (CC) in male rats estrogenized with estradiol-3-benzoate (EB). DESIGN: Prospective experimental study. SETTING: Laboratory. ANIMALS: Adult male albino rats (Holtzman strain). INTERVENTION(S): CC was given alone or in combination with EB. MAIN OUTCOME MEASURE(S): Testicular function and steroidogenic enzyme gene expression were evaluated in control versus treated groups. RESULT(S): EB after 30 days of treatment induced a rise in TUNEL-positive germ cells adversely affecting spermatogenesis with complete absence of elongated spermatids or sperms. CC alone had only a moderate effect. In contrast, CC+EB synergistically inflicted more adverse effects as apoptotic germ cells per tubule rose further. Significant down-regulation in expression of testicular steroidogenic enzyme genes StAR, p450scc, 3ß-HSD, and p450c17 was observed. In the EB-alone group, aromatase gene expression in the testis was up-regulated but reversed in brain and liver tissues. CC alone had little modulatory effect on aromatase expression. On the other hand, CC+EB countered the EB-induced rise of aromatase expression in the testis. CONCLUSION(S): The above findings indicate that CC in the presence of estrogen synergistically potentiates more adverse effects in testis, inhibiting expression of upstream steroidogenic enzyme genes and leading to disruption of steroidogenesis.

Cytoprotective effects of fruit pulp of Eugenia jambolana on H 2 O 2 -induced oxidative stress and apoptosis in rat Leydig cells in vitro.

This study was undertaken to investigate the cytoprotective effect of the fruit pulp of Eugenia jambolana (50-250 µg ml(-1) ) against the damage induced by H 2 O 2 (100 µm) exposure to Leydig cells in vitro. Cell survival with extract was found comparable to similar effects by N-acetyl-l-cysteine. H 2 O 2 -induced rise in thiobarbituric acid reactive substance formation and decline in the activity and expression of antioxidant enzymes like superoxide dismutase, catalase and glutathione-s-transferase were effectively checked. Cellular glutathione and total antioxidant capacity demonstrated significant improvement. The increase in expression of inducible nitric oxide (NO) synthase leading to NO production was successfully countered. Co-treatment of the extract helped in the down-regulation of caspase-3 and poly-ADP-ribose polymerase resulting in a significant reduction in Leydig cell apoptosis induced by H 2 O 2 . Upstream marker proteins of extrinsic (caspase-8, Fas, FasL) and intrinsic (caspase-9) pathway of metazoan apoptosis were identically down-regulated. The Bcl-2 family of proteins, though, remained unaffected. The extract also positively modulated the other marker proteins like c-Jun NH 2 -terminal kinase, p38, Akt, nuclear factor-κB, c-Fos, cellular FLICE-inhibitory protein, cyclooxygenase-2 and p53. Taken together, the above-mentioned findings establish the anti-oxidative and anti-apoptotic potency of the extract that ameliorates the H 2 O 2 -induced adverse effects on rat Leydig cells in vitro.

siRNA as a tool to delineate pathway channelization in H(2)O(2) induced apoptosis of primary Leydig cells in vitro.

Using siRNA as a tool, the channelization of pathway in H(2)O(2) induced apoptosis of primary Leydig cells was investigated in vitro. Exposure (4 h) to H(2)O(2) (250 µM) induced maximum apoptosis but affected Leydig cell viability significantly. Therefore, expression of apoptotic marker genes, caspase-8, -9, -3 and polyadenosine ribose polymerase was subsequently investigated using the same concentration post 1 h exposure. Incubation with siRNA (20 nM) either for caspase-8 or -9, inhibited their individual expressions by 55-60 % and activity, 50-55 %. The inhibition efficiency using siRNA was comparable with post- or pre-H(2)O(2) treatment of cells. Like siRNA, Eugenia jambolana (100 µg/ml) plant extract too, effectively countered over-expression of all apoptotic marker proteins. Silencing expressions of caspase 8 but not 9 through siRNA leads to a profound inhibition of caspase 3 implying that H(2)O(2) induced Leydig cell apoptosis is preferably channeled through extrinsic and later extending to other pathways.

N-acetyl-L-cysteine modulates multiple signaling pathways to rescue male germ cells from apoptosis induced by chronic hCG administration to rats.

The present study was aimed to investigate the beneficial effects of N-acetyl-L: -cysteine (NAC, 150 mg/kg bw twice/week) against testicular germ cell apoptosis in rats induced by chronic hCG administration (100 IU/rat/day for 30 days). NAC co-treatment improved serum testosterone, prevented rise in lipid peroxidation, intracellular H(2)O(2) and the activities of antioxidant enzymes in germ cells. Replenishment of intracellular GSH and total antioxidant capacity was seen. There was a marked reduction in TUNEL positive germ cells and expression of caspase-3 (p < 0.01) and PARP cleavage. Pro-apoptotic markers Fas, FasL, caspase-8 were also significantly downregulated. While Bcl-2 was fully restored, rise in Bax, caspase-9, phospho-JNK/JNK and phospho-c-Jun/c-Jun expression was significantly arrested. Anti-apoptotic phospho-Akt/Akt and NF-κB were otherwise found upregulated. Taken together, the above findings demonstrate that NAC intervention rescued the testicular germ cells from demise following chronic hCG treatment through regulation of multiple signaling mechanisms of metazoan apoptosis.

Testosterone administration to adult rats differentially modulates androgen and oestrogen receptor-α expression in reproductive organs and pituitary.

Regulation of androgen receptor (AR) and oestrogen receptor α (ERα) expression has direct bearing on the physiology of male reproductive organs. With the help of three independent tools of immunohistochemistry, western blotting and RT-PCR, AR and ER α receptor expression was examined in the testis, epididymis, prostate, seminal vesicle and pituitary of adult rats following testosterone enanthate (TE, 3 mg/100 µl of olive oil/rat per week) intervention for 15 and 30 days. TE administration reduced AR immunoexpression which coincided well with the decline in the receptor protein and transcript levels. In contrast, ERα was found overexpressed in all the organs. While weights of testis and epididymis decreased significantly, those of prostate, seminal vesicle and pituitary demonstrated an upward trend. Spermatogenesis was adversely affected with decline in number of germ cells per tubule and increased prevalence of germ cell apoptosis. Increase in serum and decrease in intra-testicular levels of testosterone were found significant (P < 0.001) in both 15 and 30 days treatment groups. Serum follicle stimulating hormone declined significantly (P < 0.001) at the end of 30 days treatment. Taken together, the above findings indicate that the testosterone intervention differentially modulates, AR ERα expression, which is associated with hypospermatogenesis and increased germ cell apoptosis.

Differential modulation of apoptotic gene expression by N-acetyl-L-cysteine in Leydig cells stimulated persistently with hCG in vivo.

The present study was designed to investigate the molecular mechanisms of NAC (150 mg/kg bw twice/week) action in vivo under repeated hCG (100 IU/rat/day) stimulation to adult rats. Leydig cell refractoriness led to a significant decline in serum testosterone and intracellular cAMP by day 30 of chronic hCG intervention which improved significantly following NAC co-administration. It inhibited the rise in lipid peroxidation, improved the activity of antioxidant enzymes along with intracellular glutathione and total antioxidant capacity in the target cells. Leydig cell apoptosis declined significantly (P<0.001) with down-regulation of upstream, Fas, FasL, caspase-8, Bax and caspase-9, JNK/pJNK and downstream caspase-3 and PARP. On the other hand, anti-apoptotic Bcl2, NF-kß, and Akt were up-regulated. Taken together, the above findings indicate that the specificity of NAC action was not restricted to regulating marker proteins in the extrinsic and JNK pathways as seen in vitro but extended to include intrinsic pathway of metazoan apoptosis as well.

Role of estrogen in the regulation of spermatogenesis in the Indian wall lizard Hemidactylus flaviviridis.

The role of estrogen in the Indian wall lizard, Hemidactylus flaviviridis during PMSG induced spermatogenesis in the regression phase and during normal spermatogenesis in the active breeding phase was investigated. Blood hormone levels demonstrated a high testosterone to estrogen ratio in the breeding and vice verse during the regressed phase. PMSG treatment (30 IU in 100 µl saline/lizard/alternate day for 30 days) during the regressed phase stimulated spermatogenesis which was associated with a significant (p<0.001) rise in plasma testosterone levels. Complete spermatogenesis with sperms was resolved in many tubular sections. However, co-administration of PMSG plus estrogen in high doses (2 µg of estradiol benzoate/alternate day) for the same period not only curtailed germ cell proliferation significantly but also induced apoptosis in germ cells. There was no significant reduction in testicular weight but sperms were found completely absent in all the tubules. Decline in the plasma testosterone was more pronounced in high compared to low estrogen treated groups. Further, low estrogen administration had little effect either on raising the plasma levels of estrogen or subsequently on spermatogenesis which was identically observed in the breeding phase too. Estrogen intervention (2 µg) in the breeding phase also profoundly suppressed spermatogenesis leading to a severe depletion in germ cells. Simultaneously, there was a significant rise in germ cell apoptosis which was associated with an up-regulation of extrinsic (caspase 8, Fas, FasL) and intrinsic (caspase 9, Bax, Bcl2) markers in these cells. Taken together, the above data indicate that the estrogen plays a key role in regulating spermatogenesis in the wall lizard retarding it during testicular quiescence and eliminating germ cells through apoptosis during the active breeding phase.

AR versus ER (α) expression in the testis and pituitary following chronic estrogen administration in adult rat.

Modulation of the testis-pituitary axis has direct relevance to the expression of androgen and estrogen receptors. Androgen receptor (AR) and estrogen receptor (ERα) expression during hypospermatogenesis after chronic estrogen administration to rats was studied in the adult testis and pituitary utilizing immunohistochemistry, western blotting, and RT-PCR. Both organs demonstrated higher AR transcriptional activity gradually increasing from 15 days (d) to 30 d of estrogen treatment. However, the AR protein as measured by either immunostaining or western blotting demonstrated a significant decline. A distinct break down of the AR protein in the pituitary into two specific bands was seen. In contrast, higher ERα transcriptional activity coincided well with the rise in protein and immunoexpression in both organs. FSH and testosterone (serum, intra-testicular testosterone) were found significantly (p < 0.001) lowered compared with raised estradiol levels. Spermatogenesis was adversely affected and was associated with a significant increase in cell apoptosis in both organs. The pituitary demonstrated a higher rate of apoptosis at the end of 30 d of estrogen treatment. Taken together, the above data indicate that chronic estrogenization to adult rats up-regulates ERα but down-regulates AR protein expression in testis and pituitary which probably has a direct association to the marked rise in cell apoptosis in these organs.

Effect of chronic oestrogen administration on androgen receptor expression in reproductive organs and pituitary of adult male rat.

Following chronic (15 or 30 days) treatment with oestradiol 3-benzoate (75 microg rat(-1) day(-1) in 100 microl of olive oil) to adult rats, androgen receptor (AR) expression was analysed simultaneously in testis, epididymis, seminal vesicle, prostate and pituitary utilising three independent tools i.e. immunohistochemistry, Western blotting and RT-PCR. All the five organs showed higher AR transcriptional activity gradually increasing from 15 to 30 days of oestrogen treatment. However, the AR protein expression either through immunostaining or Western blotting demonstrated a significant decline in all the reproductive organs. In the pituitary, on the other hand, the decline coincided with a distinct breakdown of the AR protein into two bands with increasing duration of treatment. Serum and intra-testicular testosterone levels were found significantly lowered. Spermatogenesis was adversely affected with concurrent decrease in weights of testis and accessory sex organs. Decrease in testis weight was consistent with the reduction in the number of maturing germ cells per tubule. Despite the decrease in weight, accessory sex organs like epididymis, seminal vesicle and prostate were completely devoid of any apoptotic cells which were characterised only in testis and pituitary. Seminiferous epithelium demonstrated a marked increase in the number of germ cells undergoing apoptosis. However, the rate of cell apoptosis was much higher in the pituitary than in the testis at the end of 30 days treatment. It is therefore concluded that degradation of AR protein expression after oestrogen treatment is probably directly linked to an increase in cell apoptosis both in testis and pituitary.

Release of copper from CuT 380A co-incubated with human semen and its effect on sperm function in vitro.

Release of copper and its effect on functional integrity of human sperms in vitro were assessed following co-incubation of semen with CuT 380A. After 30 min of incubation with semen, release of copper ions from CuT 380A was found to be 9.2 to 40 times higher compared to control incubations with PBS. Sperm function tests, when simultaneously performed following loss of motility in sperms (> 95%) after 120 min of copper exposure, depicted a significant (P < 0.001) reduction in sperm viability and hypo-osmotic swelling (HOS) response. However, the affected sperm populations revealed no significant alterations in other functional tests like acrosomal status or nuclear chromatin decondensation. It is therefore concluded that the high release of copper from CuT 380A drastically lowers sperm motility, viability and HOS response but only marginally affects the acrosome status or nuclear chromatin condensation in short term incubations.

Possible role of male factors in recurrent pregnancy loss.

OBJECTIVE: To evaluate various causes possibly contributing towards recurrent pregnancy loss (RPL), particularly male factors. Prospective study of 75 couples with history of RPL who were investigated for genetic, anatomic, immunological, infective and systemic causes in both partners. Functional sperm capacity was assessed by the Hypo-osmotic swelling test (HOS), Acrosomal Reaction (AR), Nuclear condensation-decondensation test (NCD) and Seminal Total Leukocyte Count (TLC) along with semen analysis. Twenty male volunteers with recently proven fertility were also included for detailed sperm morphology and sperm functions test as controls. Amongst male partners 3 (4%) had varicocele, 23 (30.6%) had infection, 1 (1.3%) immunological and 1 (1.3%) had genetic abnormality. Sperm motility, viability and sperm function tests were significantly lower in the RPL group as compared to the control group (P = 0.000). Male factor might be a possible contributing factor towards RPL. Both the partners should be evaluated and treated simultaneously in order to achieve desirable outcome.

hCG treatment raises H2O2 levels and induces germ cell apoptosis in rat testis.

The clinical significance of exogenous hCG treatment is to stimulate steroidogenesis and spermatogenesis in the testis. However, the pathogenesis of detrimental effects on the testis arising out of chronic hCG treatment is yet to be clearly ascertained. In the present study we have shown that hCG treatment (100 IU/day) to rats for 30 days raises testicular oxidative stress leading to germ cell apoptosis and impairment of spermatogenesis. The treatment raises testicular H(2)O(2) levels along with increase in lipid peroxidation and concomitant decrease in the enzymatic antioxidant activities like superoxide dismutase, catalase and glutathione-s-transferase. The rise in the number of apoptotic germ cells was associated with up regulation of Fas protein expression and caspase-3 activity in the testis. However, serum testosterone which was elevated by 15 days of hCG treatment declined to pretreatment levels by 30 days. No significant alteration in serum gonadotropins was observed. The above findings indicate that the pathogenesis of deleterious effects following chronic hCG treatment is due to increase in testicular oxidative stress with high H(2)O(2) availability leading to apoptosis among germ cells.

Estradiol treatment induces testicular oxidative stress and germ cell apoptosis in rats.

In order to understand the pathogenesis of estradiol induced effects in the seminiferous epithelium, studies were undertaken in adult rats with estradiol-3-benzoate administered for different durations. After 30 d of treatment, a significant rise in lipid peroxidation with concomitant fall in the activities of superoxide dismutase and catalase was observed. Both, serum and intra-testicular testosterone levels were found severely depleted. Seminiferous epithelium was devoid of elongated spermatids and spermatozoa by 30 d of treatment. Number of spermatocytes and round spermatids were significantly (p < 0.001) reduced. Flowcytometric analysis confirmed a drastic reduction of the haploid cell population (1c peak). Beginning from day 10 of treatment, there was a consistent rise in the number of pyknotic/apoptotic germ cells in the seminiferous epithelium. A gradual increase in Bax protein expression was observed with the duration of treatment. The shift in Bax immunostaining from the cytoplasm and nucleus of germ cells (at 10 d of treatment) to only nuclei of cells by 30 d of treatment was also noticed. By this time testicular tissue showed three-fold increase in caspase-8 enzyme activity. Viable testicular cells isolated in vitro decreased drastically subsequent to different periods of estradiol treatment. The above findings substantiate the fact that the testicular pathogenesis of estradiol benzoate treatment may be primarily because of altered reproductive hormone levels and high oxidative stress leading to germ cell apoptosis and subsequent germ cell loss in the seminiferous epithelium.

H2O2 at physiological concentrations modulates Leydig cell function inducing oxidative stress and apoptosis.

H(2)O(2) is one of the active reactive oxygen species secreted by macrophages that are seen closely aligned with Leydig cells in the testicular interstitium. The present study was initiated to investigate the role of H(2)O(2) on Leydig cell function in vitro at physiological concentrations. Significant decrease in both testosterone production (p < 0.05) and 3 beta-hydroxysteroid dehydrogenase activity (p < 0.05) in adult Leydig cells were observed even with H(2)O(2) at low concentrations (30 - 50 microM). H(2)O(2) exposure increased oxidative stress in Leydig cells with the rise in lipid peroxidation and fall in the activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT) & glutathione-s-transferase (GST). There was also a marginal increase (approximately 8%) in cell apoptosis accompanied by rise in FasL expression and caspase-3 activation. The above findings indicate that H(2)O(2) as a bio-molecule modulates Leydig cell function at or below physiological concentrations through a variety of actions like decrease in steroidogenic enzyme activity and increase in oxidative stress and apoptosis.

Germ cell death and their removal during initial stages of testicular ischemia and cryptorchidism: a comparative analysis.

Germ cell death and their removal from the seminiferous epithelium are common in the affected testis in conditions of unilateral ischemia or cryptorchidism; the similarities and differences, however, have not been studied between these two conditions. The present study was designed to examine the severity of the effect on testicular germ cells during the initial stages of both ischemia and cryptorchidism, which have significant implications on the restoration of fertility following surgical repair. Complete absence of spermatids was observed following 12 hr of ischemia as compared to 7 days of cryptorchidism. Germ cell removal in either case was in the direction of lumen to basement membrane leaving only a single layer of cells by 24 hr of unilateral ischemia as compared to 15 days of cryptorchidism. Levels of intratesticular testosterone was found lower in cryptorchidism (7 days) but not in ischemia till 24 hrs. Giant cells frequently observed in cryptorchid testis were absent in the ischemic seminiferous epithelium. There was a gradual increase in the number of apoptotic and non-viable cells; the latter was more than 95% by 24 hr of ischemia. In contrast, approximately 85% testicular cells were nonviable till 15 days of cryptorchidism. The 1c peak representing the population of haploid cells was significantly reduced in cryptorchidism (7 days), while the peak was completely abolished by 24 hr of ischemia. Rise in the levels of oxidative stress in the affected testis was observed identically during the initial stages. These findings indicate that coupled with the rise in tissue oxidative stress, the number of apoptotic/nonviable germ cells was alarmingly high (> 80%) by 15 days of cryptochidism or 24 hr of ischemia. Restoration of complete spermatogenesis following surgical repair may not be possible in such cases because of these acute adverse effects.

Apoptosis and cell removal in the cryptorchid rat testis.

In order to determine that apoptosis is responsible for large-scale germ cell elimination, we analyzed cells from cryptorchid testes both in histological sections and among those isolated in vitro. Apoptotic testicular cells during 3 to 7 days were only 8 to 30%, reaching a maximum of 80% by the end of 15 days of cryptorchidism. A similar trend was also observed with the number of dead cells. The process of large-scale germ cell removal in the initial stages was facilitated by the formation of multinucleated giant cells, which stained negative for apoptosis. Increase in oxidative stress and decrease in intratesticular testosterone was also observed. The above findings indicate that large-scale germ cell removal, at least during initial stages of cryptorchidism is not solely as a result of apoptosis. Declined intra testicular testosterone, elevated temperature and high oxidative stress following cryptorchidism probably affect cell viability and trigger a fast pace cell removal through giant cell formation.

Use of hydrogen peroxide to assess the sperm susceptibility to oxidative stress in subjects presenting a normal semen profile.

Human sperm susceptibility to oxidative stress is vital as it affects various characteristics of sperm function. In the present study, we report a simple, sensitive and quick method of assessing the capacity of the sperms to withstand increased oxidative stress. The basis for the test was derived from the fact that human sperms suspended in Ham's F-10 medium tend to lose the forward progressive motility when co-incubated with H(2)O(2) (600 microm). Replacement of the medium with seminal plasma (1: 1) was able to reduce the loss of sperm motility (40%). Retention of sperm motility in semen (0-30%) following 10 min of H(2)O(2) (600 microm) exposure was taken as the criteria for delineating the quality of sperm as poor, moderate, good and excellent types. The protocol was tested in 87 subjects presenting a normal semen profile. On the basis of this test, 44% of the semen samples were classified as poor and the rest as moderate, good or excellent. Lipid peroxidation was found higher in the sperms from the 'poor' category. Activities of superoxide dismutase and catalase were also significantly elevated in the seminal plasma of these subjects as compared with combined categories of good or excellent. The test described here can be used routinely in laboratory investigations to assess sperm susceptibility to oxidative stress in subjects presenting a normal semen profile.