Impact of moderate calorie restriction on testicular morphology and endocrine function in adult rhesus macaques (Macaca mulatta)

Abstract

We previously reported that moderate calorie restriction (CR) has minimal impact on testicular gene expression in young adult rhesus macaques, and no obvious negative impact on semen quality or plasma testosterone levels. We now extend these findings by examining the influence of CR on various aspects of the reproductive axis of older males, including 24-h circulating testosterone levels, testicular gene expression, and testicular morphology. Young adult and old adult male rhesus macaques were subjected to either 30 % CR for 5-7 years, or were fed a standard control diet. Analysis of the 24-h plasma testosterone profiles revealed a significant age-associated decline, but no evidence for CR-induced suppression in either the young or old males. Similarly, expression profiling of key genes associated with testosterone biosynthesis and Leydig cell maintenance showed no significant CR-induced changes in either the young or old animals. The only evidence for CR-associated negative effects on the testis was detected in the old animals at the histological level; when old CR animals were compared with their age-matched controls, there was a modest decrease in seminiferous tubule diameter and epithelium height, with a concomitant increase in the number of depleted germ cell lines. Reassuringly, data from this study and our previous study suggest that moderate CR does not negatively impact 24-h plasma testosterone profiles or testicular gene expression. Although there appear to be some minor CR-induced effects on testicular morphology in old animals, it is unclear if these would significantly compromise fertility.

Fig. 1

Effect of moderate CR (30 %) on daily circulating plasma T concentrations in young (a) and old (c) male rhesus macaques. Data from young adult animals were previously reported (Sitzmann et al. 2010) and show a significant difference (**P < 0.01) in minimum T levels between young controls and young CR animals (b). Differences in the present study were also detected in maximum T concentrations (*P < 0.05) between old controls and old CR animals, with the latter exhibiting higher daily levels (d). Importantly, there was no evidence for a suppressive effect of CR on plasma T levels, either in the young or old animals. (Note: to facilitate visualization of the cyclic expression patterns, SEMs have been omitted and the 24-h hormone profiles have been double plotted, with horizontal bars indicating periods of light and dark)

Fig. 2

ST diameter, epithelium height, complete germ cell line percentage, and depleted germ cell line percentage in testicular sections from young adult (a–d) and old adult (e–h) rhesus macaques. Values represent mean ± SEM. Significant effects of CR on all four morphological parameters were detected in the old animals but not in the young adults (*P < 0.05)

Fig. 3

Classification of CGL and DGL, as used in the analysis of rhesus macaque testis. Representative images of spermatogenic stages in ST sections are depicted in (a) to (f). CGLs contain all normal cell generations well represented in each cellular association (CGLNOR; a), whereas an abnormal complete germinal line (CGLABN; b) shows partial depletion in one generation of germ cells, in this case round and elongated spermatids. DGLs occur when one or more generations of germinal cell generations are absent: B spermatogonia, spermatocytes, or spermatids (DGL3; c); all round undifferentiated and elongated differentiated spermatids (DGL2; d); spermatocytes and spermatids but Sertoli cells present (DGL1; e); and germinal and Sertoli cells (fibrosed cords; DGL0; f). Abbreviations: esd elongated-differentiated spermatid, fc fibrous cord, L lumen, P pachytene spermatocyte; rsd round-undifferentiated spermatid, S Sertoli cell, sg spermatogonia. Scale bar = 50 μm

Fig. 4

Analysis of ST from calorie restricted (CR) and age-matched control (CON) rhesus macaques. Subcategories of complete germinal line (CGL) and depleted germinal line (DGL), as defined in Fig. 3, are depicted as percentages in (a) to (f)

Fig. 5

Representative semiquantitative RT-PCR gel image demonstrating expression of testicular genes in calorie restricted (CR; n = 5) or control (CON; n = 5) young adult (∼12 years old) rhesus macaques. The housekeeping gene β-actin was used as a positive control and for normalizing images for analysis. No significant differences (mean ± SEM; P > 0.05) in gene expression were observed between the treatment groups

Fig. 6

Representative semiquantitative RT-PCR gel image demonstrating expression of testicular genes in calorie restricted (CR; n = 3) or control (CON; n = 3) old adult (24–30 years old) rhesus macaques. The housekeeping gene β-actin was used as a positive control and for normalizing images for analysis. No significant differences (mean ± SEM; P > 0.05) in gene expression were observed between the treatment groups