Evaluation of the goat cellular immune response to rBtuB-Hia-FlgK peptides from Brucella melitensis.

Brucellosis is a zoonosis caused by Brucella; B. melitensis is the most prevalent species in goats and humans. Previously, three B. melitensis peptides, rBtuB-Hia-FlgK showed antigen-specific immune responses in rodent models. The goal of this study was to evaluate the goat Th1/Th2 immune response to B. melitensis peptides. Twenty-eight animals were separated into four groups and were immunized with the rBtuB-Hia-FlgK peptides cocktail, adjuvant, PBS and Rev-1 vaccine, respectively. Peripheral blood samples were collected on days 0, 15, and 80 post-inoculation. The CD4+ and CD8+ T cells proliferation, and cytokine production of the Th-1 (IL-2, IL-12, TNF-α, and IFN-γ) and Th-2 profiles (IL-4, IL-5, and IL-10) were evaluated. An increase of CD4+/CD8+ at 15 days post-vaccination was observed and continued until the 80th. In addition, the IFN-γ, TNF-α, and IL-2 mRNA expression were typically induced by the 15th day, but only IFN-γ levels were observed at day 80 post-immunization. Brucella pathogenesis is distinguished by the presence of a large amount of Th-1 cytokines. Although a reduced amount of IFN-γ in the culture supernatant was accurately detected compared with Rev-1 after 15 days, it could be influenced by the sampling schedule, as a higher cytokine production might be induced as early as the first-week post-vaccination. The results indicate that rBtuB-Hia-FlgK induced an immune response similar to the Rev-1 vaccine. The possible use of inert molecules with the unique ability to typically induce cellular response similar to attenuated vaccine represents an attractive option that should not be ruled out.

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.

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.

Simultaneous increase in germ cell apoptosis and oxidative stress under acute unilateral testicular ischaemia in rats.

Ischaemia induced germ cell apoptosis in rat testis was studied in detail to find out (i) spermatogenic stage or seminiferous epithelium region specific involvement of germ cells in apoptosis, (ii) preferential specificity of a particular germ cell type to become apoptotic and (iii) the ratio of live and dead testicular cells isolated in vitro after various period of ischaemic induction. Cell apoptosis, as observed in histological sections increased from 1 to 24 h of ischaemia. Apoptosis was not restricted to any specific germ cell type but was observed simultaneously in all the cell types in the initial hours (1-6 h) of ischaemia. No spermatogenic stage specific preference in apoptotic induction was also observed. However, as the duration of ischaemia progressed, the cell types observed to be most affected in number and morphology were the spermatids followed by spermatocytes. Centrally located tubules of testis were affected first than those located in the periphery. Overexpression of Bax staining was limited to few germ cell nuclei only. More than 95% of the germ cells in the control testis that earlier showed trypan blue dye exclusion were found stained after 12 h of ischaemia. Starting from early hours (1 h), lipid peroxidation rose proportionally with the duration of ischaemia while superoxide dismutase (SOD) and catalase activities were found decreased. Significant (p < 0.05) increase in the activities of glutathion-s-transferase and levels of hydrogen peroxide were observed after 6 h of ischaemia. These findings indicate that the physiological processes of oxidative stress have a direct linkage to the extent of germ cell apoptosis in the seminiferous epithelium.

Prepubertal human chorionic gonadotropin injection affects postpubertal germ cell maturation and androgen production in rat testis.

OBJECTIVES: To examine the effects of varying doses of prepubertal human chorionic gonadotropin (hCG) administration on postpubertal germ cell status and androgen status in rats. The long-term effects of prepubertal hCG administration on postpubertal testicular function are still debated. METHODS: Forty male prepubertal Wistar rats, aged 20 days, were divided into four equal groups. Group 1 served as the controls. Each rat in groups 2, 3, and 4 received subcutaneous hCG injections of 5 IU, 10 IU, and 50 IU, respectively, on days 20 and 30 of life. Serum testosterone levels were estimated on days 33 and 70. On day 70, the left testis of each rat was harvested for DNA flow cytometric analysis. RESULTS: The testosterone levels on day 33 progressively increased from groups 1 through 4, and the difference between any two groups was statistically significant. In contrast, the testosterone levels on day 70 were greatest in group 1 and progressively decreased in groups 2 through 4, with the lowest value in group 4. On day 70, only group 4 rats had a significantly reduced haploid cell population compared with all other groups. CONCLUSIONS: Prepubertal hCG administration adversely affects testosterone levels, and a high dose of hCG has adverse effects on the germ cell haploid cell population. A critical reevaluation of the use of hCG is required in prepubertal boys, especially with respect to the dosage.

Protective role of cyclosporine in experimental unilateral blunt testicular trauma: evaluation by 31P MR spectroscopy.

Magnetic resonance (MR) imaging is a useful tool to study the anatomy of the testis while 31P magnetic resonance spectroscopy (MRS) provides a non-invasive alternative method to demonstrate the metabolic status of testes. This study was designed to test whether the protective role of cyclosporine in experimental unilateral blunt testicular trauma (UBTT) could be assessed by 31P MRS. Male Wistar rats (n = 30) aged 20 days were randomised into group I (sham surgery), group II (UBTT) and group III (UBTT and cyclosporine for 7 days). Contralateral testicles of 5 rats from each group was evaluated by 31P MRS at 30 and 60 days of age and phosphomonoesters (PM), phosphodiesters (PD), and adenosine triphosphate (ATP) levels were measured. At 60 days of age the PM/ATP ratio was 0.32+/-0.08 in group I whereas it was 0.68+/-0.31 in the group II (p<0.05). Group III rats showed PM, PD and PM/ATP ratios similar to the controls. In conclusion, it is observed that UBTT causes contralateral testicular damage which could be prevented by short-term cyclosporine treatment and 31P MRS is an excellent modality for such an evaluation.

A short-term evaluation of semen and accessory sex gland function in phase III trial subjects receiving intravasal contraceptive RISUG.

Following the intravasal injection of a new male contraceptive RISUG (reversible inhibition of sperm under guidance) in volunteers, routine semen analysis, semen biochemistry and germ cell morphology were evaluated in comparison with the corresponding preinjection samples for a maximum period of 6 months. Sperm counts in all 25 subjects before injection varied from 45 to 120 x 10(6)/ml. Out of 25 subjects, 6 became azoospermic after 1 month, 15 after 2 months, 3 after 3 months and 1 after 4 months of contraceptive injection. The mean volume of the ejaculates was found to be less as compared to preinjection samples. Occasional sperm or sperm heads and immature germ cells were identified in only a few postinjected subjects. However, no pregnancy was reported in these subjects during the study period. Abnormal morphology found in most of the sperm, but not in the accompanying immature germ cells, may be due to a charge-related effect on the former but not on the latter cells. Neutral alpha-glucosidase, the biochemical marker for epididymis, was estimated to be significantly lower in the seminal plasma of all the postinjected subjects. On the other hand, acid phosphatase activity and fructose levels in the seminal plasma were found to be in the normal range. Based on the above findings, it is concluded that at least for the present study period, RISUG, a new male contraceptive, is effective as a partially occluding agent in the vas deferens.

Assessment of human sperm function after hydrogen peroxide exposure. development of a vaginal contraceptive.

The reactive oxygen species (ROS) that is most damaging to human spermatozoa is hydrogen peroxide. Using an artificial medium Ham's F-10, we have evaluated the effect of different concentrations of hydrogen peroxide (H(2)O(2)) on various sperm function characteristics to develop a water-based vaginal contraceptive. H(2)O(2) at 30 and 60 micro M had no effect on sperm motility. At 120 micro M of H(2)O(2), a significant reduction (90-95%) in motility was observed after 120 min. However, at 600 micro M of H(2)O(2), all the sperm were found immotile within 15 min of incubation. At much higher concentrations (1200-1500 micro M), the effect was immediate. After immobilization with H(2)O(2) (600 micro M in media), viability in sperm declined to 36% after 30 min, and was further reduced to 5% in 60 min. On the other hand, hypo-osmotic swelling response in these sperm went down drastically from 72 to 46% immediately after immobilization and there was no response at all after 60 min. In contrast, when Triton-x-100 (0.01%), a known spermicidal agent, was used in place of H(2)O(2), the immediate loss of sperm motility corresponded simultaneously with the loss of the other two functional parameters. Further, H(2)O(2) immobilized sperm revealed reduced (25-30%) nuclear chromatin decondensation as compared to 70% for the controls. A composition containing 2% H(2)O(2) in 0.9% NaCl was tested in rats as a vaginal contraceptive, depicting 100% efficacy in mating studies after 2 h of vaginal application. The findings indicate the potential of developing a water-based vaginal contraceptive using H(2)O(2) at an optimal concentration as utilized in the present study.

Effect of cyclosporine on human sperm motility in vitro.

Cyclosporine affects motility and viability of human sperm when incubated together in vitro. Sperm motility was almost reduced to nil following 10 min of incubation with cyclosporine at a concentration of 1 mg/mL. However, 200 micrograms/mL of the drug has no effect on motility and viability when tested for up to 60 min under standard laboratory conditions. Cyclosporine effect on sperm was both dose and time dependent. Sperm sensitivity and susceptibility to cyclosporine even to lower doses increased significantly following withdrawal of bovine serum albumin from the incubating medium. Compared to untreated controls, lactate dehydrogenase was estimated higher by more than 2 to 4 times in the sperm-free incubating media, suggesting an altered membrane porosity in the affected spermatozoa.