Capacitation-dependent concentration of lipid rafts in the apical ridge head area of porcine sperm cells

Lipid architecture of the plasma membrane plays an important role in the capacitation process of the sperm cell. During this process, an increase in membrane fluidity takes place, which coincides with a redistribution of cholesterol to the apical region of the head plasma membrane and subsequently an efflux of cholesterol. Cholesterol is also a major player in the formation of lipid rafts or microdomains in the membrane. Lipid rafts favour specific protein-protein interactions by concentrating certain proteins in these microdomains while excluding others. In this study, we investigated the organization of lipid rafts during in vitro capacitation of boar sperm cells. We report on the presence of the lipid raft-specific proteins caveolin-1 and flotillin-1 in sperm cells. Capacitation induced a change in membrane distribution of these proteins. Lipid analysis on detergent-resistant membranes (DRMs) of sperm cells indicated that capacitation induces a lipid raft concentration rather than a disintegration of lipid rafts, because the total amount of lipid in the DRM fraction remained unaltered. Using a proteomic approach, we identified several major DRM proteins, including proteins involved in capacitation-dependent processes and zona pellucida binding. Our data indicate that sperm raft reorganization may facilitate capacitation-specific signalling events and binding to the zona pellucida.     

A combination of interleukin-6 and its soluble receptor impairs sperm motility: implications in infertility associated with endometriosis

BACKGROUND: We previously reported that the level of interleukin (IL)-6 is increased in the peritoneal fluid of women with endometriosis. This study was undertaken to assess the effects of IL-6 and soluble IL-6 receptor (sIL-6R) on in vitro sperm motility. METHODS: Sperm (n = 20) were cultured with IL-6 or sIL-6R, or with a combination of both. After 24 h cultures, sperm motility was evaluated using a computer-assisted semen analysis system. Gene and protein expressions of IL-6, IL-6 receptor (IL-6R), and glycoprotein 130 (gp130) were examined in sperm by RT-PCR analysis and western blot analysis. RESULTS: Addition of IL-6 or sIL-6R individually to the culture media had no affect on sperm motion. However, adding a combination of IL-6 and sIL-6R dose-dependently reduced the percentage of motile and rapidly moving sperm. Adding anti-IL-6R antibody abolished these adverse effects. Sperm expressed the gp130 gene and protein, but not IL-6 or IL-6R. CONCLUSIONS: A combination of IL-6 and sIL-6R may be associated with gp130 expressed in the sperm and reduce sperm motility. IL-6 and sIL-6R may contribute to the pathogenesis of endometriosis-associated infertility.     

Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations

Although various genetic factors have been implicated in human male infertility, the causative genes for the different types of idiopathic male infertility have not been elucidated. Protamines, which are the major DNA-binding proteins in the sperm nucleus, package the DNA into the sperm head. Analysis of the human protamine-1 (PRM1) and -2 (PRM2) gene sequences in 226 sterile male patients and in 270 proven-fertile male volunteers revealed four single nucleotide polymorphisms (SNPs) in the PRM1 coding region, which did not cause any amino acid substitutions, and one SNP in the PRM2 gene, which produced translation termination. We also observed one SNP in the 3' non-coding region of the PRM1 gene, and two SNPs within the intron of the PRM2 gene. The prevalence of these SNPs was similar in both infertile patients and in proven-fertile volunteers, except that the c248t alteration in the PRM2 gene induced a nonsense codon under conditions of heterozygosity in one infertile patient. Although the PRM1 and PRM2 genes are highly conserved, the single SNP in the PRM2 gene that induces translation termination may result in male infertility due to haploinsufficiency of PRM2.     

Sperm ubiquitination positively correlates to normal morphology in human semen

BACKGROUND: Global ubiquitination in human semen has been found to negatively correlate with standard semen parameters, indicating that ubiquitination can be considered a marker of poor semen quality. However, the inclusion of all semen components in the analysis may be misleading on the biological significance of ubiquitination of sperm cells. We have recently demonstrated the variable presence of bodies of different size, with the highest concentration in oligoasthenoteratozoospermia. The purpose of the present study was to evaluate the relationship between ubiquitination and standard semen parameters, after distinguishing between ubiquitinated sperm and bodies in each sample. METHODS: Ubiquitination was evaluated by flow cytometric sperm ubiquitin tag immunoassay (SUTI) in sperm samples from 45 subjects. Semen analysis was performed according to WHO (1999) guidelines. RESULTS: When only ubiquitinated sperm were considered, a positive correlation with number, motility and normal morphology was found. When correlation was evaluated considering the percentage of ubiquitinated bodies, a negative correlation was found with good semen quality. CONCLUSIONS: Results indicate that the negative correlations previously found between global semen ubiquitination and parameters of semen analysis are mainly driven by components other than sperm. The positive correlation between sperm ubiquitination and good quality parameters suggests a previously unrecognized role for sperm ubiquitination.     

Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa.

The levels of superoxide dismutase (SOD), a highly specific scavenging enzyme for superoxide anion radicals (O2-), and lipid peroxide produced by oxygen free radicals were measured in human seminal plasma and spermatozoa. Seminal plasma contained 366.8 +/- 20.9 U/ml (mean +/- SE) of SOD activity. SOD activity in human spermatozoa showed a significant correlation to the number of motile spermatozoa, while the activity in seminal plasma did not relate to the sperm concentration or motility. The lipid peroxide concentration in seminal plasma was 6.22 +/- 0.46 nmol/ml and had no significant relationship to sperm concentration or motility. The malondialdehyde (MDA) concentration in spermatozoa was significantly related to the number of immotile spermatozoa. A decrease in the motility of spermatozoa incubated in medium without seminal plasma was observed after 120 min, while the MDA concentration of the spermatozoa increased. Addition of exogenous SOD (400 U/ml) to the sperm suspension significantly decreased this loss of motility and the increase of the MDA concentration. These data suggest a significant role for SOD in sperm motility. It seems that lipid peroxidation of human spermatozoa may cause loss of motility and that SOD may inhibit this lipid peroxidation. These results suggest that SOD may have a possible clinical application in the use of spermatozoa for in-vitro fertilization (IVF) or artificial insemination.     

Sperm morphological assessment based on strict criteria and in-vitro fertilization outcome.

One-hundred-and-twenty-three in-vitro fertilization (IVF) cycles were analysed in order to clarify the influence of strictly normal morphology (SNM) of spermatozoa on IVF outcome. SNM was defined using strict criteria according to Kruger with our modifications. The IVF cycles studied were divided into three groups: %SNM less than 12% (13 cycles), 12 less than 40% (68 cycles), greater than or equal to 40% (42 cycles). The cleavage rates per oocyte were higher in the groups with 12-40% and greater than or equal to 40% of %SNM than in the group with %SNM less than 12%. The embryo transfer rate per cycle increased with increasing %SNM. The overall pregnancy rate per cycle increased with increasing %SNM (7.7% in %SNM less than 12%, 22.1% in 12-40% of %SNM, and 40.5% in %SNM greater than or equal to 40%). The ongoing pregnancy rate per cycle also increased with increasing %SNM (7.7% in %SNM less than 12%, 14.7% in 12-40% of %SNM, and 31.0% in %SNM greater than or equal to 40%). The miscarriage rate was lower in %SNM greater than or equal to 40% (23.5%) than in 12-40% of %SNM (33.3%). It was suggested that %SNM is a good predictor of IVF outcome.     

Andrology: Factors influencing the outcome of in-vitro fertilization with epididymal spermatozoa in irreversible obstructive azoospermia

Microsurgical epididymal sperm aspiration (MESA) and in-vitrofertilization (IVF) were found to offer limited opportunityfor fatherhood to 45 men with obstructive azoospermia, due principallyto poor embryo implantation. Adequate sperm preparations wereobtained in 46/50 treatment cycles (92%), with the best motilityfound in the caput epididymis in 89% of cases. The mean fertilizationrate was 11.2% and fertilization occurred in 23 cycles (50%),with embryo transfer arising from 12/26 men with was aplasia(CAV), 4/9 with genital tract obstruction (EV) and 7/11 withirreversible vasectomy (VV). The overall implantation rate waslow, 8.7% per embryo transfer (11.7% per 2-3 embryo transfers)and was not improved by Fallopian transfer. There were two pregnancies(4% per cycle), both in the EV group where embryo formationand implatation (2/4, 50% per cycle) were optimum even thoughsperm preparations were paradoxically inferior to the CAV andVV groups. The spermatozoa retrieved in the two successful EVcycles were appreciably blood contaminated. Analysis of the21 failed embryo transfers showed delayed fertilization in 10cycles, cystic fibrosis (CF) mutation or familial disease in7/12 CAV men and the VV men were older (P<0.001). A pregnancywhich miscarried arose from a case of Young's syndrome, a carrierof CF mutation DF508. Male factors could thus be implicatedin the high embryo wastage of MESA cycles and might also beinfluencing implantation in other IVF procedures. Where feasible,male reconstructive surgery is preferable unless fertilizationcan be improved, possibly by speedier retrieval techniques orby permitting sperm capacitation in vitro, but probably moreeffectively by micro-assisted insemination.     

Fertility of ejaculated and testicular megalohead spermatozoa with intracytoplasmic sperm injection

In this study the fertility and outcome of intracytoplasmic sperm injection (ICSI) using megalohead spermatozoa from the ejaculates and testicles was evaluated. Seventeen males with megalohead and pinhead sperm forms in their ejaculate were studied in 22 cycles. A high number of sperm heads without tails and abundant round spermatid forms were commonly observed. Round-headed spermatozoa were seldom accompanied by these severely abnormal spermatozoa. The majority of megalohead spermatozoa were observed to have multiple tails, were predominant in the sample, and were used for ICSI. Ejaculated megalohead spermatozoa were used for ICSI in 15 cycles, while testicular spermatozoa were used in seven cycles where there were no vital spermatozoa or spermatozoa of low vitality in the ejaculate. The same abnormal morphology was observed in the testicles as in the ejaculated spermatozoa in the same males. Mean (+/- SD) low motility 4.7 +/- 5.6% and sperm count (3.8 +/- 4.19 x 10(6)) were common findings in these severely teratozoospermic patients. A low fertilization rate (43.2%) was achieved by using megalohead sperm forms (group I, n = 17) in comparison with the control group (60.2%) which had zero normal sperm morphology according to strict criteria (group II, n = 30) (P <0.01). Furthermore, a low pregnancy rate (9.1%) was obtained in the megalohead sperm group in comparison with the control group (40%) (P <0.05). Low fertilization and pregnancy rates may be due to a high incidence of chromosomal abnormalities from severely defective spermatozoa in the ejaculate. Couples should be counselled and warned about possible low fertilization and pregnancy rates with ICSI when only pinhead and megalohead forms with a high number of sperm heads without tails are present in the ejaculate.     

An algorithm to predict pregnancy in assisted reproduction

BACKGROUND: Male fertility potential cannot be measured by conventional parameters for the assisted reproduction technique; ICSI. This study determines the relationship between testicular and ejaculated sperm mitochondrial (mt) DNA deletions, nuclear (n) DNA fragmentation, and fertilization and pregnancy rates in ICSI. METHODS: Ejaculated sperm were obtained from 77 men and testicular sperm from 28 men with obstructive azoospermia undergoing ICSI. Testicular sperm were retrieved using a Trucut needle. mtDNA was analysed using a long PCR. The alkaline Comet assay determined nDNA fragmentation. RESULTS: Of subjects who achieved a pregnancy (50%) using testicular sperm, only 26% had partners' sperm with wild-type (WT) mtDNA. Of pregnant subjects (38%) using ejaculated sperm, only 8% had partner sperm with WT mtDNA. In each, the successful group had less mtDNA deletions and less nDNA fragmentation. There were inverse relationships between pregnancy and mtDNA deletion numbers, size and nDNA fragmentation for both testicular and ejaculated sperm. No relationships were observed with fertilization rates. An algorithm for the prediction of pregnancy is presented based on the quality of sperm nDNA and mtDNA. CONCLUSION: In both testicular and ejaculated sperm, mtDNA deletions and nDNA fragmentation are closely associated with pregnancy in ICSI.     

Testicular sperm extraction in azoospermic men submitted to bilateral orchidopexy

BACKGROUND: This study was carried out to evaluate whether bilateral orchidopexy represents a poor or good prognostic factor in azoospermic men undergoing testicular sperm extraction (TESE). METHODS: One hundred and seven presumed non-obstructive azoospermia (NOA) patients, according to conventional clinical parameters (volume of testis, FSH, clinical history) were submitted to testicular biopsy with TESE. Thirty men (28%) had a history of bilateral orchidopexy for cryptorchidism. RESULTS: Normal spermatogenesis or mild hypospermatogenesis was diagnosed in 12/30 ex-cryptorchid patients and in 7/77 presumed NOA patients (P = 0.0004). Conversely, pure Sertoli cell-only syndrome or complete maturation arrest was found in 10/30 ex-cryptorchid patients and in 48/77 presumed NOA patients (P = 0.0094). In 53/107 patients (49.5%), TESE allowed a positive sperm retrieval. At least one spermatozoon was observed in 22/30 ( approximately 73%) ex-cryptorchid patients and in 31/77 ( approximately 40%) presumed NOA patients (P = 0.0026). A large number of spermatozoa (equivalent to an obstructive pathology) were retrieved in 13/30 ex-cryptorchid and in 10/77 presumed NOA patients (P = 0.001). A history of bilateral orchidopexy in presumed NOA patients correlates positively for the chance of retrieving testicular spermatozoa (odds ratio 3.8; 95% confidence interval 1.41-10.21; P = 0.008). CONCLUSIONS: Although bilateral cryptorchidism is usually considered a testicular secretive dysfunction, TESE permits retrieval of a large number of spermatozoa in almost 40% of cases. Our data suggest the existence of congenital or acquired obstructive anomalies of the seminal ducts in azoospermic orchidopexed men.