Abdorrahman S. Alghamdi

Seminal DNase Frees Spermatozoa Entangled in Neutrophil Extracellular Traps

Abstract Insemination always stimulates neutrophil migration into the female reproductive tract (FRT), which eliminates excess spermatozoa and bacterial contaminants introduced by the breeding process. However, the presence of neutrophils in the FRT at the time of semen deposition has been shown to result in sperm-neutrophil binding that reduces motility and fertility. Although the binding and trapping mechanism has not been determined, seminal plasma (SP) was found to include a protein factor or factors that reduced sperm-neutrophil binding and improved fertility of sperm inseminated in the presence of neutrophils. Although DNase has been shown to be present in the SP of different species and has been associated with improved fertility in bulls, the mechanism(s) explaining this association and the paradox of DNA-packed cells being associated with DNase have remained unresolved. We demonstrate that sperm-activated neutrophils extrude their DNA, which in turn traps sperm cells and hinders their motility (and ultimately may hinder sperm transport to the fertilization site). DNase activity present in the SP digests the extruded DNA and frees entangled spermatozoa, which in turn may allow more spermatozoa to reach the oviduct, and explains at least one mechanism by which SP increases the rate of fertility. The ability of SP proteins to suppress neutrophil activation in the presence of spermatozoa did not render neutrophils incapable of combating bacteria, demonstrating that SP proteins are highly selective for suppressing neutrophils activated by spermatozoa, but not by bacteria.

Nitric Oxide Levels and Nitric Oxide Synthase Expression in Uterine Samples from Mares Susceptible and Resistant to Persistent Breeding-induced Endometritis

Problem:  Breeding‐induced endometritis (BIE) in the mare is resolved by 36 hr after insemination in resistant mares. However, 10–15% susceptible broodmares fail to do so because of impaired uterine contractility between 7 and 19 hr after exposure to seminal or bacterial challenge, which reduces their fertility.

Uterine secretion from mares with post-breeding endometritis alters sperm motion characteristics in vitro

Uterine secretion was collected from five normal mares during estrus by the use of a tampon. In subsequent estrus cycles, mares were inseminated with 1 x 10(9) spermatozoa from a stallion of known fertility, and uterine secretion was collected randomly at 6, 12, and 24 hours after insemination. All mares had negative endometrial cytology before insemination. At the time of uterine secretion sampling, semen was collected from two stallions and extended with Kenneys extender to a concentration of 50 x 10(6) spermatozoa/mL. Extended semen was diluted 2:1 with uterine secretion; semen extender; and centrifuged uterine secretion (noncellular). Samples were kept at room temperature and sperm motion characteristics (corrected motility (CMOT), progressively motile spermatozoa (PMS), and mean path velocity (MPV) were evaluated using a computer-assisted semen analyzer every 40 minutes for a total of 4 hours. Sperm motion characteristics of spermatozoa were significantly better when incubated in semen extender compared to uterine secretion (P < 0.05). The CMOT and PMS were significantly better in uterine secretion collected before, compared to after AI with the lowest values observed in samples collected at 12 hours after breeding (P < 0.05). Sperm motion characteristics of spermatozoa incubated in centrifuged uterine secretion was only slightly suppressed compared to spermatozoa incubated in semen extender, suggesting that the altered motion characteristics were mostly due to the presence of polymorphonuclear neutrophils (PMNs) in the samples. It was concluded from this study that spermatozoa can survive in inflamed uterine secretion, but that sperm motion characteristics in vitro are altered.

Equine Sperm-Neutrophil Binding

ABSTRACT When mares are inseminated repeatedly, protein molecules from the seminal plasma (SP) prevent sperm-neutrophil binding and reduced fertility. The molecule(s) responsible for sperm-neutrophil binding is not known and the identification of beneficial SP proteins is complicated by their large numbers and abundant variation. We examined several important aspects of sperm-neutrophil binding to ultimately facilitate the identification and isolation of the molecule(s) responsible. First, we raised anti-equine P-selectin antibodies to determine the involvement of this adhesion molecule in sperm-neutrophil binding. While these antibodies identified equine P-selectin, they did not inhibit sperm-neutrophil binding. However, acrosome-reacted equine sperm expressed a molecule similar to the ligand recognition unit of P-selectin. Second, we attempted to characterize SP protein binding to equine sperm and gauge their affinity. Biotinylated SP proteins were incubated with fresh sperm, washed over a viscous medium, electrophoresed, and probed with avidin. Several SP proteins bound to sperm with a strong affinity to withstand these treatments. This finding may prove valuable for future attempts to identify and characterize specific SP molecules. Lastly, we compared the secretions from male sex organs/glands on sperm motility, sperm-neutrophil binding, and their protein profile. We expected fewer proteins from individual organs/glands, which would facilitate isolation and identification of target molecules. While each secretion had a varying effect on motility and sperm-neutrophil binding, the protein profile was as complex as that seen in whole SP, indicating that collection of proteins from individual sources will not facilitate this work. Together, these experiments answer several important questions related to sperm-neutrophil binding, sperm-SP proteins interaction, and the complexity of the SP proteome.

Response to a Letter to the Editor by Dr. Kahn

Dear Editor, I have read Dr. Firdous Ahmad Kahn’s letter and concerns regarding discrepancies of two research reports from my laboratory conducted 8 years apart. I have also responded to a similar letter by Dr. Kahn to the journal Reproduction in Domestic Animals. Dr. Kahn’s major concern was based on a his estimation of a substantial difference between the two studies in the total amount of NO in uterine secretion of mares with induced breeding-induced endometritis. Dr. Kahn also pointed out that one of the publications incorrectly reported total NO in mM, which is expressing a concentration and not total amount. He made the assumption that the correct unit should be mmol and based his calculations on this. In response to Dr. Kahn’s letter, I contacted the corresponding authors of the two reports, Drs. Alghamdi and Woodward. Following a thorough review of all original data and recalculations of the results, it was concluded that calculation of the total amount of NO in the two studies was performed by the use of similar mathematical methods and that both studies should have reported the total amount of NO in umol. An unfortunate error was made in the AJRI report from 2005 by incorrectly labeling the total amount in mM rather than the correct unit (umol) – not mmol as assumed in Dr. Kahn’s letter. This brings the results in the two reports much closer than expressed in the letter to the editor, but there are still some differences between the studies. We would, however, caution against attempts to make a direct comparison of the results between the two reports, as they were not conducted under the same conditions. For example, slightly different methods were used to induce the inflammation, and while one of the studies reported NO at 6 hr as well as the percent change over time, the other study reported NO at 13 hr only, and no identical time points were evaluated among the two experiments. None of the experiments was conducted with the objective of establishing normal or absolute values of NO accumulation in the uterus following induced inflammation, but rather to compare the relative accumulation of NO in the uteri of mares susceptible to breeding-induced endometritis with resistant mares. Both studies arrived to similar conclusions, supporting the hypothesis that susceptible mares have an abnormal accumulation of NO following breeding-induced endometritis. We truly regret the unfortunate error in labeling total amount of NO with an incorrect unit in one of the reports, and hope this response will help in avoiding further misunderstandings and misinterpretation of the data. Best regards,