Trypanosomes and mammalian sperm: one of a kind?

Flagellar-mediated motility is an indispensable function for cell types as evolutionarily distant as mammalian sperm and kinetoplastid parasites, a large group of flagellated protozoa that includes several important human pathogens. Despite the obvious importance of flagellar motility, little is known about the signalling processes that direct the frequency and wave shape of the flagellar beat, or those that provide the motile cell with the necessary environmental cues that enable it to aim its movement. Similarly, the energetics of the flagellar beat and the problem of a sufficient ATP supply along the entire length of the beating flagellum remain to be explored. Recent proteome projects studying the flagella of mammalian sperm and kinetoplastid parasites have provided important information and have indicated a surprising degree of similarities between the flagella of these two cell types.

Serum hyperglycosylated human chorionic gonadotropin to predict the gestational outcome in in vitro fertilization/intracytoplasmic sperm injection pregnancies

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted by the placenta in early pregnancy. Decreased H-hCG levels have been associated with abortion in spontaneous pregnancy. We retrospectively measured H-hCG and dimeric hCG in the sera of 87 in vitro fertilization patients obtained in the 3 weeks following embryo transfer and set the results in relation to pregnancy outcome. H-hCG and dimeric hCG were correlated (r(2) = 0.89), and were significantly decreased in biochemical pregnancy (2 microg/l and 18 IU/l, respectively) compared to early pregnancy loss (22 microg/l and 331 IU/l) and ongoing pregnancy (32 microg/l and 353 IU/l). Only H-hCG tended to discriminate between these last two groups.

Anti-Müllerian hormone and inhibin B as predictors of pregnancy after treatment by in vitro fertilization/intracytoplasmic sperm injection

OBJECTIVE: To evaluate anti-Müllerian hormone (AMH) as a marker of reproductive outcome after IVF/intracytoplasmic sperm injection (ICSI). DESIGN: Longitudinal study. SETTING: University hospital. PATIENT(S): Two hundred seventy-six consecutive women undergoing IVF/ICSI. INTERVENTION(S): Ovarian stimulation, oocyte retrieval, IVF, ICSI, embryo transfer, AMH, and inhibin B determinations in serum and follicular fluid (FF). MAIN OUTCOME MEASURE(S): The AMH and inhibin B concentrations in 276 matched FF/serum pairs have been determined. Different outcome groups have been compared and set in relation to the oocyte count, morphological parameters, and steroid hormone levels. RESULT(S): The concentrations of AMH and inhibin B in both serum and FF were significantly higher in the group of women who became pregnant in the corresponding treatment cycle than in those who did not conceive. Positive correlations were observed between serum inhibin B concentrations and embryo morphology (r = 0.126, 95% confidence interval 0.026-0.284). Serum and FF AMH or inhibin B correlated positively with the oocyte count and negatively with the pretreatment cycle day 3 FSH level and the total administered gonadotropin dose. CONCLUSION(S): The AMH and inhibin B levels on the day of oocyte retrieval are correlated to reproductive outcome.

Intravaginal and intracervical application of seminal plasma in in vitro fertilization or intracytoplasmic sperm injection treatment cycles--a double-blind, placebo-controlled, randomized pilot study

OBJECTIVE: To evaluate whether intravaginal application of seminal plasma at the time of follicle aspiration in IVF or intracytoplasmic sperm injection treatment cycles has the potential to increase pregnancy rate. To calculate the number of patients needed to achieve significance in a multicenter trial. DESIGN: Double-blind, placebo-controlled randomized pilot study. SETTING: University department of gynecological endocrinology and reproductive medicine. PATIENT(S): One hundred sixty-eight patients undergoing IVF or intracytoplasmic sperm injection treatment. INTERVENTION(S): Cryopreserved seminal plasma from the patient's partner or sodium chloride (placebo) was injected into the cervix and the posterior fornix of the vagina just after follicle aspiration. MAIN OUTCOME MEASURE(S): Clinical-pregnancy rate. RESULT(S): One hundred sixty-eight patients agreed to participate in the study. Participation was limited to one treatment cycle. Thirty-one patients (18%) were excluded from the study, mainly as a result of canceled embryo transfers. Seventy patients received placebo, and 67 received seminal plasma. The clinical-pregnancy rate was 25.7% (18/70) in the placebo group. The clinical-pregnancy rate in the seminal plasma group reached 37.3% (25/67), corresponding to a relative increase of 45%. CONCLUSION(S): Even though significance was not reached in this pilot study, the data suggest that seminal plasma has the potential to improve pregnancy rate. It is estimated that around 450 patients need to be recruited to reach significance in a multicenter study.

DNA methylation patterns at the IGF2-H19 locus in sperm of Swiss Landrace and Swiss Large White boars

DNA methylation patterns at the IGF2-H19 locus were investigated in sperm DNA from Swiss Landrace (SL) and Swiss Large White (LW) boars. The putative IGF2 differentially methylated regions (DMR) 0, 1 and 2, a quantitative trait nucleotide (QTN) region in the intron 3 and a CpG island in the intron 4 of the IGF2 gene as well as three regions around porcine CTCF binding sites within the H19 differentially methylated domain (DMD) were selected for the DNA methylation analysis. In both breeds putative IGF2 DMR0, 1, 2 and H19 DMD were hypermethylated. Significant differences in DNA methylation content were found between the two breeds in the two DMD regions proximal to the H19 gene. The IGF2 QTN region and the CpG island in the IGF2 intron 4 were hypomethylated in sperm DNA of both breeds. The methylation analysis revealed significantly more methylated CpG sites in the intron 4 of sperm from the LW breed than in that from SL. No difference was found in global DNA methylation between the two breeds. These results indicate differences in DNA methylation patterns between breeds and it remains to be established whether variation in DNA methylation patterns impacts on phenotypic traits.

In search of epigenetic marks in testes and sperm cells of differentially fed boars

In search of transmittable epigenetic marks we investigated gene expression in testes and sperm cells of differentially fed F0 boars from a three generation pig feeding experiment that showed phenotypic differences in the F2 generation. RNA samples from 8 testes of boars that received either a diet enriched in methylating micronutrients or a control diet were analyzed by microarray analysis. We found moderate differential expression between testes of differentially fed boars with a high FDR of 0.82 indicating that most of the differentially expressed genes were false positives. Nevertheless, we performed a pathway analysis and found disparate pathway maps of development_A2B receptor: action via G-protein alpha s, cell adhesion_Tight junctions and cell adhesion_Endothelial cell contacts by junctional mechanisms which show inconclusive relation to epigenetic inheritance. Four RNA samples from sperm cells of these differentially fed boars were analyzed by RNA-Seq methodology. We found no differential gene expression in sperm cells of the two groups (adjusted P-value>0.05). Nevertheless, we also explored gene expression in sperm by a pathway analysis showing that genes were enriched for the pathway maps of bacterial infections in cystic fibrosis (CF) airways, glycolysis and gluconeogenesis p.3 and cell cycle_Initiation of mitosis. Again, these pathway maps are miscellaneous without an obvious relationship to epigenetic inheritance. It is concluded that the methylating micronutrients moderately if at all affects RNA expression in testes of differentially fed boars. Furthermore, gene expression in sperm cells is not significantly affected by extensive supplementation of methylating micronutrients and thus RNA molecules could not be established as the epigenetic mark in this feeding experiment.

Effect of cancer chemotherapy on the frequency of minisatellite repeat number changes in human sperm

The objective of this study was to determine whether cancer chemotherapy induces detectable mutations in DNA of the human germline and whether minisatellite repeat number changes can be used as a sensitive indicator of genetic damage in human sperm caused by mutagens. We compared the mutation frequencies in sperm of the same cancer patients pre- and post-, pre- and during, or during and post-treatment. Small pool polymerase chain reaction (SP-PCR) (DNA equivalent to approximately 100 sperm) and Southern blotting techniques were used to detect mutations and quantify the frequency of repeat number changes at the minisatellite MS205 locus. One pre- and one post-treatment semen sample was obtained from each Hodgkin's disease patient treated with either: (1) a regimen without alkylating agents, Novantrone, Oncovin, Vinblastine, and Prednisone (NOVP), 4 patients; (2) a regimen containing alkylating agents, Cytoxan, Vinblastine, Procarbazine, and Prednisone (CVPP)/Adriamycin, Bleomycin, DTIC, CCNU, and Prednisone (ABDIC), 2 patients; and (3) a regimen containing alkylating agents, Mechlorethamine, Oncovin, Procarbazine, and Prednisone (MOPP), 1 patient. One pre- and one during treatment semen sample from each of two Hodgkin's disease patients treated with Adriamycin, Bleomycin, Vinblastine, and Dacarbazine (ABVD) were obtained. One during and one post-treatment semen sample from a Hodgkin's disease patient treated with NOVP were also obtained. At least 7900 sperm in each sample were screened for the repeat number changes at the MS205 locus by multi-aliquots of SP-PCR. The mutation frequencies of pre- and post-treatment for the four patients treated with NOVP were 0.22 and 0.18%; 0.24 and 0.16%; 0.35 and 0.28%; and 0.19 and 0.18%. With CVPP/ABDIC, they were 0.22 and 0.23%; and 0.94 and 0.98% for the two patients and with MOPP they were 0.79 and 1.14%. The mutation frequencies of pre- and during treatment with ABVD were 0.09 and 0.07%; and 0.34 and 0.27% for the two patients. The mutation frequencies of during and post-treatment with NOVP for one patient were 0.31 and 0.25%. A statistically significant increase in mutation frequency was only found in the patient treated with MOPP. According to the time of samples collected after or during treatment and the above results, we conclude that there is no effect of NOVP and CVPP/ABDIC regimens on the mutation frequency in spermatogonia. The spermatocytes are not highly sensitive to chemotherapy agents compared to spermatogonia at the minisatellite MS205 locus. MOPP treatment may increase the mutation frequency at the MS205 locus in spermatogonia. ^

Effect of Multiple Freezing of Stallion Semen on Sperm Quality and Fertility

To increase the efficiency of equine semen, it could be useful to split the artificial insemination dose and refreeze the redundant spermatozoa. In experiment I, semen of 10 sires of the Hanoverian breed, with poor and good semen freezability, was collected by artificial vagina, centrifuged, extended in INRA82 at 400 × 106 sperm/mL, and automatically frozen. After this first routinely applied freezing program, semen from each stallion was thawed, resuspended in INRA82 at 40 × 106 sperm/mL, filled in 0.5-mL straws, and refrozen. These steps were repeated, and sperm concentration was adjusted to 20 × 106 sperm/mL after a third freezing cycle. Regardless of stallion freezability group, sperm motility and sperm membrane integrity (FITC/PNA-Syto-PI-stain) decreased stepwise after first, second, and third freezing (62.3% ± 9.35, 24.0% ± 15.4, 3.3% ± 4.34, P ≤ .05; 29.6% ± 8.64, 14.9% ± 6.38, 8.3% ± 3.24, P ≤ .05), whereas the percentage of acrosome-reacted cells increased (19.5% ± 7.59, 23.9% ± 8.51, 29.2% ± 6.58, P ≤ .05). Sperm chromatin integrity was unaffected after multiple freeze/thaw cycles (DFI value: 18.6% ± 6.6, 17.2% ± 6.84, 17.1% ± 7.21, P > .05). In experiment II estrous, Hanoverian warmblood mares were inseminated with a total of 200 × 106 spermatozoa of two stallions with either good or poor semen freezability originating from the first, second, and third freeze/thaw cycle. First-cycle pregnancy rates were 4/10, 40%; 1/10, 10%; and 0/10, 0%. In conclusion, as expected, sperm viability of stallion spermatozoa significantly decreases as a consequence of multiple freezing. However, sperm chromatin integrity was not affected. Pregnancy rates after insemination of mares with refrozen semen are reduced.

Importance of sperm transition proteins demonstrated in mice carrying Tnp1- and Tnp2-null alleles

Chromatin condensation within the nucleus of developing spermatids involves replacement of histones by transition proteins, which are in turn replaced by protamines. The importance of transition proteins in the complex process of spermiogenesis has, to date, been only speculative. This study sought to investigate the extent to which transition proteins are essential or have redundant functions by characterizing sperm produced in mice expressing all combinations of Tnp-null alleles. Results from breeding trials of 8 weeks duration revealed that, on average, wildtype males produced about 14 offspring whereas TP2 and TP1 single-knockout males produced about 8 and 1 offspring, respectively, demonstrating their subfertility. Genotypes with less than two Tnp wildtype alleles, as well as double-knockout mutants, were completely infertile. Sperm from males with impaired fertility had poor progressive motility, heterogeneous chromatin condensation, incompletely processed protamine 2 and head and tail abnormalities. Generally, as the number of Tnp-null alleles increased so did the severity of abnormalities. However, specific morphological abnormalities were associated with the absence of an individual TP. Studies which sought to identify possible root causes for abnormalities in thiol-rich sperm structures revealed no differences in thiol content or sulfhydryl oxidation status within the nucleus but nuclei and tails from single-knockout mutants were severely disrupted following thiol reduction. Binding of fluorescent dyes to DNA was normal in sperm recovered from caput but abnormal in cauda epididymal sperm from TP1 knockouts and infertile double mutants. Injection of cauda epididymal sperm from double knockouts into oocytes produced very few offspring; however, after injection with testicular sperm, the efficiency was no different from wildtype. These results suggest DNA structural alterations or degradation during epididymal transport of sperm resulting in a diminished capacity of the paternal DNA of these sperm to produce offspring. The overall importance of transition proteins for normal chromatin condensation and production of fertile sperm has been demonstrated. Furthermore, identification of specific morphological abnormalities associated with the absence of an individual transition protein provides new evidence that the proteins are not completely redundant and each fulfills some unique function. ^

Ocean acidification impacts on sperm mitochondrial membrane potential bring sperm swimming behaviour near its tipping point

Broadcast spawning marine invertebrates are susceptible to environmental stressors such as climate change, as their reproduction depends on the successful meeting and fertilization of gametes in the water column. Under near-future scenarios of ocean acidification, the swimming behaviour of marine invertebrate sperm is altered. We tested whether this was due to changes in sperm mitochondrial activity by investigating the effects of ocean acidification on sperm metabolism and swimming behaviour in the sea urchin Centrostephanus rodgersii. We used a fluorescent molecular probe (JC-1) and flow cytometry to visualize mitochondrial activity (measured as change in mitochondrial membrane potential, MMP). Sperm MMP was significantly reduced in delta pH -0.3 (35% reduction) and delta pH -0.5 (48% reduction) treatments, whereas sperm swimming behaviour was less sensitive with only slight changes (up to 11% decrease) observed overall. There was significant inter-individual variability in responses of sperm swimming behaviour and MMP to acidified seawater. We suggest it is likely that sperm exposed to these changes in pH are close to their tipping point in terms of physiological tolerance to acidity. Importantly, substantial inter-individual variation in responses of sperm swimming to ocean acidification may increase the scope for selection of resilient phenotypes, which, if heritable, could provide a basis for adaptation to future ocean acidification.