Positive effects of prolonged caloric restriction on the population of very small embryonic-like stem cells - hematopoietic and ovarian implications

Abstract

Background: Low calorie intake, or calorie restriction (CR) without malnutrition, has been demonstrated in several animal species, including mice, to increase both median and maximum lifespan as well as delay reproductive senescence. Our previous work demonstrated a positive correlation between life span and the number of very small embryonic-like stem cells (VSELs) in long living Laron dwarf mice. These animals have very low levels of circulating insulin-like growth factor 1 (IGF-1) in peripheral blood (PB), maintain higher numbers of hematopoietic stem cells (HSPCs) in bone marrow (BM), and display prolonged fecundity compared with wild type littermates. Since CR lowers the level of IGF-1 in PB, we become interested in the effect of CR on the number of VSELs and HSPCs in BM as well as on the morphology of ovaries and testes.

Methods: In our studies four-week-old female and male mice were subjected to CR by employing an alternate-day ad libitum feeding diet for a period of 9 months.

Results: We observed that mice on CR had a higher number of BM-residing VSELs than control mice fed ad libitum. These changes correlated with higher numbers of HSPCs in BM, spleen, and peripheral blood (PB) as well as with an increase in the number of primordial and primary follicles in ovaries. At the same time, however, no changes were observed in the testes of mice under CR.

Conclusion: We conclude that CR positively affects the pool of VSELs in adult tissues and explains the positive effect of CR on longevity.

Keywords: Bone marrow; Calorie restriction; Longevity; Ovaries; Testes; VSELs.

Figure 1

Increases in the number of stem cells in the BM of 10-month-old female and male C57B1/6 mice on CR compared with controls fed AL. The number of Sca-1⁺Lin^–CD45^– VSELs (left panels) and Sca-1⁺Lin^–CD45⁺ HSPCs (right panels) in the BM of mice on CR or of control mice fed AL (6 mice/group).

Figure 2

The number of clonogenic BFU-E and CFU-GM in hematopoietic tissues of 10-month-old female (panel A) and male (panel B) C57B1/6 mice on CR or fed AL. Mononuclear cells from bone marrow (A), peripheral blood (B), or spleen (C) from mice (6 animals/group) were plated in vitro in methylcellulose cultures to evaluate the number of clonogenicerythroid BFU-E and myeloid CFU-GM progenitors. The number of colonies grown from mice fed AL is shown as 100%.

Figure 3

Effect of CR on morphology of ovaries and testes. Panel A. Ovaries of 10-month-old C57B1/6 mice fed AL (a, c, and e) or on CR (b, d, and f). a and b. In both groups, primary (PF), preantral (PAF), and antral (AF) follicles and corpus luteum (CL) are visible. We also observed primordial follicles and a greater number of atretic follicles (red arrowheads) in the CR group (b) than in the AL-fed group (a). Hematoxylin + eosin staining. Magnification: x100. Inset in panel b represents primordial follicles in an ovary from a mouse on CR. Magnification, x400. c-f. Immunohistochemical reaction for growth hormone receptor (GHR) expression in ovaries of mice fed AL (c and e) or on CR (d and f). High expression of GHR in ad libidum(AL)-fed mice was noted in granulosa cells (red arrow), preantral and antral follicles and zona pellucida (asterisk), ovarian surface epithelium (black arrowhead), and blood vessel endothelium (BV, c and e). By contrast, expression of GHR in CR mice was found only in a few granulosa cells (red arrow) of Graffian follicles (GF), and no expression of GHR was observed in granulosa cells of preantral (PAF), primary (PF), and primordial (PRF) follicles (d and f). Magnification: panels c and d, x100; panels e and f, x200. Representative images are shown. Panel B. The number of follicles at different stages of development in ovaries of 10 month-old female C57B/6 mice on CR compared with controls fed AL. The higher number of primordial, primary, preantral and atretic follicles in ovaries of mice on CR than in ovaries of mice fed ad libidum. P < 0.01. Panel C. Testes of 10-month-old C57B1/6 mice fed AL (panels a and c) or on CR (panels b and d). Magnification: panels a and b, x100; panels c and d, x200. PAS staining. Representative images are shown.