Modeling the effect of cigarette smoke on hexose utilization in spermatocytes

Abstract

We set out to determine whether the addition of an aryl hydrocarbon receptor (AHR) antagonist has an effect on glucose/fructose utilization in the spermatocyte when exposed to cigarette smoke condensate (CSC). We exposed male germ cells to 5 and 40 μg/mL of CSC ± 10 μmol/L of AHR antagonist at various time points. Immunoblot expression of specific glucose/fructose transporters was compared to control. Radiolabeled uptake of 2-deoxyglucose (2-DG) and fructose was also performed. Spermatocytes utilized fructose nearly 50-fold more than 2-DG. Uptake of 2-DG decreased after CSC + AHR antagonist exposure. Glucose transporters (GLUTs) 9a and 12 declined after CSC + AHR antagonist exposure. Synergy between CSC and the AHR antagonist in spermatocytes may disrupt the metabolic profile in vitro. Toxic exposures alter energy homeostasis in early stages of male germ cell development, which could contribute to later effects explaining decreases in sperm motility in smokers.

Keywords: GLUT12; GLUT9a; cigarette smoke; fructose; spermatocytes.

Figure 1.

Effects of cigarette smoke condensate (CSC) ± aryl hydrocarbon receptor (AHR) antagonist on hexose uptake. A, 2-Deoxyglucose (2-DG) uptake by spermatocytes treated with the dimethyl sulfoxide (DMSO; vehicle control), CSC (40 μg/mL), AHR antagonist, or CSC + AHR antagonist, as indicated. N = 2 experiments in triplicate. B, Fructose uptake by spermatocytes treated with the DMSO (vehicle control), CSC, AHR antagonist, or CSC + AHR antagonist, as indicated. ** indicates P = .01 (analysis of variance [ANOVA] compared to DMSO). N = 2 experiments in triplicate. All values are expressed as mean ± standard error of the mean (SEM).

Figure 2.

Glucose transporter (GLUT) expression profile of spermatocytes. A, Western blot of previously (GLUT 5, 8, and 9a) and newly (GLUT 12) described GLUT transporters present in murine spermatocytes. Cigarette smoke condensate (CSC) alone does not affect whole cell protein expression among GLUT 5, 8, 9a, and 12; however, GLUT 9a and GLUT 12 begin to decrease with concurrent exposure to AHR antagonist. All Western immunoblots represent multiple determinations (>3). B, Quantification of fold change (expressed as standard error of the mean [SEM]) among GLUT 5, 8, 9a, and 12 normalized to actin. Comparisons between control (dimethyl sulfoxide [DMSO]) and CSC + antagonist for 24 hours for GLUT 9A and GLUT 12 approach significance.

Figure 3.

Effect of cigarette smoke condensate (CSC) on adenosine triphosphate (ATP) production in spermatocytes. When examining the percentage change in ATP production in CSC-exposed spermatocytes compared to control (dimethyl sulfoxide [DMSO]), there is a significant dose-dependent effect. With both 20 and 40 μg/mL CSC, the spermatocyte ATP production decreased significantly (P < .05) compared to 5 μg/mL.

Figure 4.

Spermatogenesis. Type A spermatogonia undergoing mitosis and either replenishing pool of germ cells or differentiating into type B spermatogonia (basal compartment of seminiferous tubule). Meiosis I and II: preleptotene spermatocytes cross blood testis barrier (BTB) into adluminal compartment where meiosis II produces haploid round spermatids. Spermiogenesis and spermiation: haploid round spermatids undergo spermiogenesis, which is defined by the formation of the acrosome, and are released from the seminiferous epithelium into tubules. Reprinted from Lie PPY, Cheng Y, Mruk, DD. Coordinating cellular events during spermatogenesis: a biochemical model. Trends in Biochemical Sciences. 2009;34:366-373 with permission from Elsevier.