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Sperm mRNA transcriptional profiles can be used to evaluate male fertility, yet differences between species in sperm attributes and packaging require adjustments in sperm RNA isolation protocols. The objective was to optimize RNA isolation methodology for fresh, frozen, and extended ejaculates, and epididymal sperm of stallions. Additionally, a protocol for RNA isolation from testis biopsies was established. Separation of sperm from somatic cells was critical for assuring the isolation of sperm-specific RNA. The highest purity was obtained by centrifuging ejaculates and epididymal sperm at 200 x g for 30 min through a 40% Equipure silanized silica particle solution. Sperm RNA isolation was more efficient with TRIzol reagent than with a spin-column based method; it resulted in 2 microg of total RNA per 100 x 10(6) sperm. To evaluate RNA quantity and quality, we used a NanoDrop spectrophotometer and Agilent Bioanalyzer. A protocol for reverse transcriptase PCR with equine primers for PRM2 and PTPRC genes was developed to determine sperm RNA contamination with genomic DNA or RNA from somatic cells. By these methods, hybridization- and sequencing-quality RNA was isolated from 11 samples of stallion sperm. Stallion testis biopsy with a 14 gauge 22 mm deep biopsy needle yielded approximately 12 microg of good quality total RNA, and could serve as an alternative to excision surgery for sample procurement. Compared to RNA isolation from testis, the sperm required advanced processing and RNA quality control. The described methodologies provided a foundation to establish functional genomic studies of stallion fertility., ((c) 2010 Elsevier Inc. All rights reserved.)

Objective: To determine whether or not sperm function parameters are altered in male partners of couples with a history of idiopathic recurrent pregnancy loss (RPL)., Design: In comparison with proven fertile volunteers, sperm function parameters like hypo-osmotic swelling (HOS), acrosomal status (AS), and nuclear chromatin decondensation (NCD) were assessed in vitro from male partners of couples with a history of idiopathic RPL., Setting: Infertility clinic and andrology laboratory at National Institute of Health and Family Welfare., Patient(s): Male partners of couples with a history of idiopathic RPL and proven fertile male volunteers (control)., Intervention(s): Standard semen analysis, assessment of sperm morphology, and sperm function with tests such as HOS, AS, and NCD., Main Outcome Measure(s): Sperm paameters, such as HOS, AS, and NCD, were assessed in semen samples from RPL in comparison with the proven fertile control group., Result(s): Semen samples from the idiopathic RPL group showed below normal test scores in 57.1% of the cases for all three sperm parameters. The highest aberration (83% of cases) in sperm attributes was observed in NCD, followed by AS (45.7%) and HOS (42.9%). In contrast, abnormality in sperm morphology was limited to 5.7% of the cases. Subnormal sperm function is directly proportional with subnormal sperm motility (<50%) in 23% of the cases. Even in semen samples with normal sperm motility, sperm function scores were below normal in 31.4% of the RPL group., Conclusion(s): Reduction in test scores of sperm function, like HOS, AS, and NCD, in male partners of couples with idiopathic RPL suggests that sperms with altered or lowered functional competencies, if they fertilize the oocytes, may lead to the development of an unsustainable embryo resulting in early pregnancy loss. Normal sperm motility does not always ensure normal sperm function scores.

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.

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.

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.

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.

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.

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.