Molecular Chaperone HSPA2 Distribution During Hyaluronic Acid Selection in Human Sperm

Abstract

During fertilization, sperm hyaluronidase activity is essential for spermatozoa to successfully penetrate the hyaluronic acid-enriched extracellular matrix of the cumulus cells. Since molecular chaperones, as the heat shock protein A2, are typically involved in bringing hyaluronic acid receptors to the cell surface, here we evaluated the presence and spatial location of HSPA2 on human spermatozoa based on its hyaluronic acid binding capacity. This study included 16 normozoospermic sperm samples from volunteering donors. The location of HSPA2 was studied in cells before and after 1-h incubation under capacitating conditions, as well as in spermatozoa selected according to their ability of binding to hyaluronic acid. Our results showed no significant differences in HSPA2 immunofluorescent cells before and after 1 h of incubation in capacitating conditions. Nevertheless, after hyaluronic acid selection, the percentage of HSPA2-labelled cells increased significantly, indicating that the interaction with hyaluronic acid may induce the unmasking of HSPA2 epitopes. Furthermore, after swim-up and hyaluronic acid selection, spermatozoa presented a highly immunostained equatorial band with a homogeneous fluorescence throughout the acrosomal region. This distribution has been previously suggested to have important implications in male fertility. Noteworthy, a homogeneous fluorescence among the acrosomal region with a more intense labelling at the apical region was observed only in hyaluronic acid bound sperm cells, which may be associated with primary gamete recognition. Our findings suggest that the hyaluronic acid selection technique and HSPA2 biomarker should be considered candidates to complement the classic seminal analysis before recommending an appropriate assisted reproduction technique.

Sperm Adhesion Molecule 1 (SPAM1) Distribution in Selected Human Sperm by Hyaluronic Acid Test

“…Therefore, as a novelty, our results emphasize that human sperm require the presence of SPAM1 throughout the sperm head (P1) to properly contact the cumulus-oocyte matrix. These results support the usefulness of the HA test as a natural sperm selector [ 19 , 29 ].…”

“…In this way, the sperm with HA receptors, upon encountering the SpermSlow medium, were trapped in the junction area of the two drops and received the name of mature sperm (MS), whereas sperm without these receptors were able to swim through the droplet and were termed immature (IS). This methodology was performed following previous protocols of our group [ 29 ].…”

IZUMO1 Receptor Localization during Hyaluronic Acid Selection in Human Spermatozoa

“…The tests to evaluate the effectiveness of this new technique included the presence and distribution of different biomarkers. Previous studies of our group conducted after HA selection showed a significant increase in cells labeling HSPA2, a heat shock protein A2 involved in bringing hyaluronic acid receptors to the cell surface [29] and the requirement of the presence of SPAM1 (sperm adhesion molecule 1) throughout the sperm head to properly contact the cumulus-oocyte matrix [30]. Both studies show the localization of two proteins involved in the primary recognition of gametes; however, the location of biomarkers implicated in the next steps of fertilization is unknown.…”

“…Based on previous studies from our group [29,30], the semen samples were processed to obtain spermatozoa under the following conditions (Figure 1): noncapacitated sperm (NCS), one-hour-capacitated sperm (CS1), mature and immature sperm selected by HA after one-hour capacitation (MS1 and IS1), and induced acrosome reaction from one-hourcapacitated sperm (ARS1). Mature and immature groups were termed by Cayli et al [13].…”

“…After 10 min of incubation at 37 • C under oil (FertiCult TM Mineral Oil, FertilPro, Beemen, Belgium), the sperm with HA receptors were trapped in the junction area of the two drops and received the name mature sperm (MS1), whereas sperm without these receptors were able to swim through the SpermSlow medium droplet and were termed immature (IS1). This methodology was performed following previous protocols of our group [29,30].…”