No abstract

Evidence suggests that porcine spermatozoa bind to the endometrium lining after entering the uterus. In previous studies, it was verified that this binding is specific to porcine uterine epithelial cells (UEC). Here, we present trials aimed at identifying the engaged binding mechanism. Several interactions of spermatozoa with the female reproductive tract are known to be lectin mediated. The respective sugar ligands are species specific for the oviductal as well as zona pellucida surface membrane. Therefore, we hypothesised that sperm-endometrium interactions are mediated by lectins, too. To block possibly existing sugar moieties on the sperm surface, we pre-incubated spermatozoa with lectins selected for their strong binding to viable spermatozoa [wheat germ agglutinin WGA, succinylated WGA (sWGA), concanavalin A (ConA)] and subsequently undertook co-incubation studies using an established cell culture model from primary porcine uterine epithelial cells. All trials were carried out in modified Dulbecco’s Modified Eagle’s Medium containing 20% serum (D20) because the UEC monolayer proved to react sensitive to PBS as well as to semen extender. Pre-trials confirmed that in D20 sperm vitality remained unaffected and lectin binding efficiency did not differ compared with PBS. The sperm-rich fraction of the ejaculate from 3 German Landrace boars was collected in D20 and washed by centrifugation. The pellet was resuspended in D20 and the concentration adjusted to 100 × 106 spermatozoa mL–1. Then, the samples were incubated with 1 of the 3 lectins (10 µg mL–1) for 15 min at 37°C. As a control, 1 aliquot of the sperm suspension was incubated without lectins. After another washing step, the pellet was resuspended in D20. For co-incubation with UEC, 500 µL of lectin-incubated sperm was released onto a UEC monolayer and the binding activity observed under a phase contrast microscope. The binding density was quantified by area under view and compared to results from the control incubation with untreated sperm. Images (2 repeats/boar and lectin) were divided into fields of 61.6 µm2 and the fields with and without sperm were counted. For statistical analysis, a Tukey Test was carried out. Sperm treated with WGA and sWGA, both binding to glucosamine molecules, showed significantly reduced binding activity to the UEC (P < 0.05), whereas ConA-treated and untreated sperm bound in the dense pattern as shown in previous trials. The results indicate that the sperm-UEC binding mechanism is mediated by glucosamine moieties on the sperm surface, which interact with matching lectins placed on the UEC membrane surface. Investigations for further confirmation of the suggested binding mechanism between porcine sperm and the uterine epithelial layer are ongoing. Supported by IMV Technologies, L’Aigle, France.

Many important goals have been reached by reproductive biotechnologies such as sperm cryopreservation and sperm sorting. However, these procedures have often been recognized to induce changes in sperm that can affect their quality. The detrimental influence of sperm handling during reproductive biotechnologies is attributed to the dilution, and the consequent decrease in the concentration of seminal plasma, changes in temperature, buffers, osmotic stress, and mechanical forces. These stresses can induce sperm death and nonlethal modification such as membrane destabilization, which can have the features of premature capacitation. These sperm changes can alter the delicate and specific sequence of events that prepare sperm for the fertilizing event. An extensive knowledge of these handling induced changes is fundamental in order to minimize them, thus optimizing sperm survival and function. Many studies have been focused on methods effective in protecting and stabilizing sperm surface such as the use of cholesterol, antioxidants and, in particular, the exploitation of the protective effects of seminal plasma in order to improve the quality of spermatozoa subjected to reproductive biotechnologies. The workshop speakers will present an overview on the current research and the future perspective on this challenging topic.During their journey from the testis to the fertilization site, spermatozoa must undergo a series of changes in order to attain the ability to fertilize the oocyte. These changes include modifications of the sperm membrane by addition and later removal of seminal plasma (SP) proteins. Application of new sperm biotechnologies, such as sperm cryopreservation and flow cytometric sex-sorting, involve a substantial dilution or removal of SP. These procedures can alter sperm surface configuration and induce capacitation-like changes leading to a decrease in sperm fertilizing ability. Attempts to improve the outcome of these biotechnologies involve the restoration of SP to the sperm sample. The beneficial effect of SP on the spermatozoa is variable and depends upon different factors, including the source of SP, the processing of SP and the point of addition of SP to the sample. This variability can be partly overcome by the application of isolated SP proteins. In boars, most of the SP proteins belong to the sperm adhesin family. Recent studies are pointing to the PSP-I/PSP-II sperm adhesin as one of the protective factors of the SP that could be used as an additive to post-sorting collection medium. This protective effect could be related to the ability of the PSP-I/PSP-II heterodimer to hold the spermatozoa in a non-capacitated state. Further studies are needed to get a better knowledge of the effect of whole SP or its proteins in biotechnologically treated sperm functionality. (Supported by SEN-ECA (GERM 04543/07) and MINECO (AGL2012-39903), Spain.) WS 1.3 Sperm surface changes caused by assisted reproductive technologies and the influence of protective factors: how well do we speed or slow sperm readiness...