Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa

Abstract

Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaglyceroporins (AQPs)-7 and -9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light-and electron microscope levels with several commercial primary antibodies explored AQP-location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate); un-processed, extended, chilled and frozen-thawed.Although differences in WB-and ICC-labelling were seen among antibodies, AQP-7 was conspicuously located over the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid-piece and principal piece domains in ejaculated spermatozoa.AQP-9 was mainly localized over the sperm head both in caudal and ejaculated spermatozoa.While unaffected by chilling (+5ºC), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP-7-, but not that of AQP-9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP-9 but relocated AQP-7 towards the acrosome. AQP-7 but not AQP-9 appears as a relevant marker for non-empirical studies of sperm handling.

Aquaporins in the male reproductive tract and sperm: Functional implications and cryobiology

“…As Vicente‐Carrillo et al. () found AQP7 in connecting, mid‐ and principal pieces of boar spermatozoa, not only in frozen‐thawed but also in fresh sperm, more studies are required to address to which extent the nature of the sample underlies those localization differences.…”

“…AQP7 is found in round and elongated spermatids, in the tail of testicular sperm and in the mid‐piece of ejaculated sperm of mice, rats, humans, bulls, boars and stallions (Bonilla‐Correal et al., ; Ishibashi et al., ; Prieto‐Martínez, Morató et al., ; Saito et al., ; Vicente‐Carrillo, Ekwall, Álvarez‐Rodríguez, & Rodríguez‐Martínez, ; Yeung et al., , ; Table ).…”

“…In addition, although Vicente‐Carrillo et al. () found AQP9 in the head of epididymal and ejaculated boar sperm, this protein was suggested to play a marginal role during chilling, freezing and thawing procedures (Table ). Thus, even though more research is required to confirm the presence of AQP9 in the sperm of other mammalian species, current evidence indicates that, if present, its role is not as important as that of the other AQPs.…”

“…In a very interesting paper, Vicente‐Carrillo et al. () showed that exposure of boar sperm to freezing medium induced changes in AQP7 localization, as AQP7 staining was observed in the sperm head after freeze‐thawing. Boar and bull spermatozoa with higher relative AQP3 and AQP7 content have also been suggested to be more resilient to withstand cryopreservation procedures, which, as aforementioned, includes the osmotic shock of freezing media (Prieto‐Martínez, Morató et al., ; Prieto‐Martínez et al., ).…”

Cryotolerance of Stallion Spermatozoa Relies on Aquaglyceroporins rather than Orthodox Aquaporins

“…Even if ubiquitous, the presence of AQPs varies between cell types and species. In mammalian spermatozoa, AQP1 has been identified in porcine [3]; AQP3, AQP7 and AQP11 in equine [4], porcine [5,6], murine [7][8][9], human [10,11] and bovine species [12,13]; AQP8 has been found in mouse [8] and human spermatozoa [10,11]; and AQP9 is present in boar spermatozoa [14]. In the male gamete, AQPs are involved in the regulation of cell volume and osmotic balance, which are crucial for spermatogenesis [15] and post-ejaculation events, including the activation of sperm motility upon ejaculation and sperm adaptation to the female environment (reviewed in [2]).…”