Time course of hypo-osmotic swellings of human spermatozoa: evidence of ordered transition between swelling subtypes

Abstract

The hypo-osmotic swelling test (HOST or HOS test) usually takes into consideration the total HOS response value with no emphasis either on the value of the response subtypes or the response evaluation time. This study investigated the time course of HOS responses and analysed their physiological relevance. Raw semen spermatozoa and Percoll washed spermatozoa were used in the experiment. The morphological changes in the sperm tail were monitored by incubating the spermatozoa in the hypo-osmotic solution for 16 different time periods. The HOS reactive spermatozoa and the type of HOS reaction (swelling subtypes) of the samples subjected to different duration of treatment were identified under a phase contrast microscope. Also the fate of individual spermatozoa in a hypo-osmotic environment were monitored for 30 min. In spermatozoa exposed to a hypo-osmotic solution, the motility lasted usually less than 2 min and motility characteristics were uniquely different from that of the spermatozoa under iso-osmotic conditions. The HOS response development was permanent but the motility loss due to hypo-osmotic shock was reversible up to 1 min of incubation. There was an indication of ordered transition among the HOS swelling subtypes apparently initiating with subtype b destined to c, d, e, f and g. Further, the subtypes a and g showed gradual decrease and increase, respectively, while subtype b showed abrupt initial increase and then gradual decrease. Transition from b to g could be direct or via one or more than one subtypes. Ultrastructure based analysis indicated that HOS response subtypes are the apparent reflection of the differences in the cytoskeletal assembly of the sperm tail and thus may be identifying different physiological variants in the sperm population. These results indicate that shorter incubation is essential to document the kinetics of various HOS responses but the conventional HOS test misses these important HOS features because of lengthy incubation. Since the time course of ordered transition of HOS responses will vary more than the total HOS response in semen of different aetiologies, the importance of HOS response subtypes and response evaluation time should be taken into consideration when applying HOS test.

Relação da taxa de gestação com sêmen bovino congelado e testes de avaliação espermática in vitro

“…A baixa resposta ao teste HO após a criopreservação provavelmente foi ocasionada por danos na cauda dos espermatozóides durante o processo de congelamento/ descongelamento, o que diminuiu a capacidade desses espermatozóides de reagirem ao aumento de volume celular, em resposta à baixa osmolaridade (Hossain et al 1998), ou em decorrência das condições de estoque e manuseio do sêmen (Correa & Zavos, 1994). Além disso, o próprio meio de congelamento (hiperosmótico) pode acarretar ruptura de membrana, pois o estresse osmótico pode induzir, além de alterações nas estruturas das membranas, mudanças nas estruturas microtubulares da cauda (Correa & Zavos, 1994).…”

Sole Use of Sucrose in Human Sperm Cryopreservation

“…The Makler chamber was used for motility evaluation [6,7] and motility was graded following WHO criteria [6,7]. Hypo-osmotic swelling (HOS) test was applied as an indirect method to determine the viability and membrane integrity of sperm [14][15][16]. This was accomplished by adding 5 mL of sample to 50 mL of hypo-osmotic solution (HS).…”

“…This was accomplished by adding 5 mL of sample to 50 mL of hypo-osmotic solution (HS). The HS was constituted with citrate (7.35 g=L) and fructose (13.5 g=L) in distilled water to obtain 150 mmol=kg osmolarity [14,15,17]. The sperm was mixed thoroughly with HS and incubated for 20 minutes at 37 C before microscopy examination for hypo-osmotic swelling [14,16,17].…”

The viability of spermatozoa

“…[13] ont montr6 que les diff6rents types de gonflement-incurvation flagellaire r6pondent ~ une cin6tique bien d6finie. Ainsi, la r6ponse initiale apparait ~ partir de 25 secondes d'incubation, toujours selon le type b (Figure 1), caract6ris6 par un discret gonflement ~ l'extr6mit6 du flagelle.…”