Capacitation of ram spermatozoa promotes changes in energy metabolism and aquaporin 3 and is affected by individual testosterone variations

Abstract

Background: Recently, the metabolic pathways involved in energy production and the role of aquaglyceroporins in capacitation-associated events have been studied in humans and mice. However, little is known about these in ram spermatozoa.

Objective: The present study investigated bioenergetic and aquaglyceroporin 3 variations during in vitro capacitation of ram spermatozoa. In addition, differences in testosterone levels between males were examined to determine their influence on capacitation-like changes.

Materials and methods: Spermatozoa obtained from nine rams (ejaculates = 36) were incubated for 180 min in three different media (control, capacitating, and aquaglyceroporin-inhibitor media) at 38.5°C. At 0 and 180 min of incubation in each medium, sperm viability, kinetics, chlortetracycline patterns, adenosine triphosphate concentration, lactate excretion (final subproduct of glycolysis), and immunolocalization of aquaporin 3 were evaluated.

Results: The increment of the capacitated spermatozoa-chlortetracycline pattern and the hyperactivated-like movement characterized by the highest curvilinear velocity and amplitude of lateral head displacement and the lowest linearity was only recorded after 180 min in the capacitating medium. At this time and conditions, adenosine triphosphate content and lactate excretion decreased, whereas the aquaglyceroporin 3 location in the midpiece and principal piece increased compared to 0 min. Such changes were not observed in the control medium over time. Incubation in the aquaglyceroporin-inhibitor medium for 180 min reduced drastically sperm motility and adenosine triphosphate content compared to the other media. Testosterone analysis revealed a significant individual variability, which was also present in all sperm parameters evaluated. Furthermore, testosterone was negatively correlated with adenosine triphosphate content but positively correlated with lactate excretion levels, sperm viability, motility, capacitated sperm-chlortetracycline pattern, and aquaglyceroporin 3 immunolabeling in the midpiece and principal piece.

Conclusion: Despite individual differences, capacitation of ram spermatozoa increases adenosine triphosphate consumption, energy metabolism, and aquaglyceroporin 3 location in the midpiece and principal piece, which seems to be related to the acquisition of hyperactivated-like motility. Furthermore, testosterone levels may serve as a valuable tool to select those males with a greater sperm metabolism rate and fertilizing capacity.

Keywords: aquaglyceroporins; capacitation; hyperactivated motility; sperm metabolism; testosterone variations.

FIGURE 1

Sperm viability and acrosomal loss during the incubation period in different media. The mean percentages of all males ± SEM in the saline (SM), capacitating (CAP) and inhibitor (INHIB) media at 0 and 180 min are shown. Different letters show significant differences (p < 0.05) among incubation media and times for each parameter assessed.

FIGURE 2

Capacitated (CTC CAP) and acrosome reacted (CTC AR) CTC patterns during the incubation period. (A) Mean percentages of CTC patterns of all males ± SEM in the saline (SM), capacitating (CAP) and inhibitor (INHIB) media at 0 and 180 min. Different letters show significant differences (p < 0.05) among incubation media and times in each CTC pattern. (B) Mean percentages of CTC AR pattern in each male ± SEM during the incubation period in CAP medium. (C) Mean percentages of CTC CAP pattern in each male ± SEM during the incubation period in CAP medium. ^*Indicate significant differences (p < 0.05) among incubation times for each male.

FIGURE 3

Sperm kinetic descriptors during the incubation period in saline (SM), capacitating (CAP), and inhibitor (INHIB) media. (A) Total motility; (B) VCL: curvilinear velocity; (C) LIN: linearity and (D) ALH: lateral head displacement. Data are expressed as means of all males ± SEM. Different letters show significant differences (p < 0.05) among incubation media and times for each parameter assessed.

FIGURE 4

ATP content of ram sperm during the incubation period. (A) Mean values of ATP for all males ± SEM in the saline (SM), capacitating (CAP) and inhibitor (INHIB) media at 0 and 180 min. Different letters show significant differences (p < 0.05) among incubation media and times. (B) Mean values of ATP in each male ± SEM during the incubation period in CAP medium. ^*Indicate significant differences (p < 0.05) among incubation times for each male.

FIGURE 5

Lactate excretion in ram sperm during the incubation period. (A) Mean values of lactate for all males ± SEM in the saline (SM), capacitating (CAP) and inhibitor (INHIB) media at 0 and 180 min. Different letters show significant differences (p < 0.05) among incubation media and times. (B) Mean values of lactate excretion in each male ± SEM during the incubation period in CAP medium. ^*Indicate significant differences (p < 0.05) among incubation times for each male.

FIGURE 6

Immunolocalization of aquaglyceroporin 3 (AQP3) in ram spermatozoa. Green fluorescence shows AQP3 located in the acrosome (ACR), midpiece (MP), and principal piece (PP). Cell nuclei were stained blue with Hoechst 33342. (A) Two patterns can be distinguished: sperm with the absence of AQP3 (NF) and sperm with AQP3 located at the midpiece and acrosome region. (B) Two patterns can be distinguished: sperm with AQP3 located in the midpiece and principal piece, while in others only in the midpiece. (C) Three patterns can be distinguished: sperm with the absence of AQP3 (NF), sperm with AQP3 located only at the acrosome region, and sperm with AQP3 located at the acrosome, midpiece, and principal piece. (D) Two patterns can be distinguished: sperm with AQP3 located only at the acrosome region and sperm with AQP3 located at the midpiece and principal piece.

FIGURE 7

Percentage of ram sperm showing aquaglyceroporin 3 (AQP3) immunolabeling in the midpiece (IP), principal piece (PP), and acrosome (A) during the incubation period. (A) Mean percentages of all males ± SEM in the saline (SM), capacitating (CAP) and inhibitor (INHIB) media at 0 and 180 min. Different letters show significant differences (p < 0.05) among incubation media and times in each sperm region. (B) Mean percentages for each male ± SEM during the incubation period in CAP medium. ^* Indicate significant differences (p < 0.05) among incubation times for each male.

FIGURE 8

Testosterone levels for each male (mean ± SEM). Different letters denote significant differences (p < 0.05) among males.

FIGURE 9

Possible roles of aquaglyceroporin 3 (AQP3) in ram sperm physiology and capacitation. Partially designed by Biorender.