Caloric restriction in female reproduction: is it beneficial or detrimental?

Figure 1a indicates that some metabotropic neurons in hypothalamus enable to regulate HPO axis. Corticotropin releasing hormone (CRH) neurons in hypothalamus of adult female rats directly inhibit Kiss1^ARC and Kiss1^AVPV through CRH receptors [10]. The orexigenic neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons in ARC are negative to HPO axis. Padilla et al. [11] discovered that AgRP neurons in mice give inhibitory innervation to Kiss1^ARC and Kiss1^AVPV, but they do not give any neurotransmitter or neuropeptide to GnRH neurons. Although GnRH neurons in female rodents express both stimulatory NPY Y4 receptors and inhibitory Y1 receptors, the latter is responsible for the major effect of NPY peptide on GnRH neurons [12, 13]. GnRH neurons in adult rats also express inhibitory NPY Y5 receptors [14]. The anorexigenic pro-opiomelanocortin (POMC)/cocaine- and amphetamine-regulated transcript (CART) neurons in ARC are positive to HPO axis. In female mice, the excitatory effect of POMC neurons to GnRH neurons is predominantly mediated by the POMC-cleaved product α-melanocyte stimulating hormone (α-MSH), which excites GnRH neurons via both melanocortin receptor 3 (MC3R) and MC4R [12]. However, α-MSH inhibits CRH neurons via MC4R [15]. The experiment in female mice found that AgRP peptide, which is an antagonist of melanocortin receptors, attenuates the MC4R-mediated activation on GnRH neurons [16]. Interestingly, POMC neurons in female mice negatively innervate NPY/AgRP neurons and this innervation is enhanced by estradiol (E₂) [17]. It has been discovered that CART postsynaptically depolarize Kiss1^ARC and GnRH neurons in female rats [18]. Collectively, in normal energy status, CRH neurons and ARC NPY/AgRP neurons inhibit HPO axis while ARC POMC/CART neurons activate HPO axis.

Leptin is an adipocyte-derived anorexigenic factor. The stimulatory effect of leptin on HPO axis is dominant at hypothalamic level. Leptin directly activates Kiss1^ARC in mice, sheep and guinea pigs. It is summarized that leptin deficiency in mice decreases not only ARC kiss1 mRNA level but also the amounts of Kiss1^AVPV [19]. Although GnRH neurons do not express leptin receptors (LepRs) [19‐21], leptin in rodents can indirectly stimulate them through the neurons in hypothalamic premammillary nucleus (PMV) [22]. Generally, ARC POMC/CART neurons express facilitatory LepR while ARC NPY/AgRP neurons express inhibitory LepR [15]. Indeed, leptin can exert female-specific stimulatory effect on GnRH neurons via CART in adult rats [14]. However, strong evidences from laboratory rodents indicate that NPY-Y1/Y5 receptor signaling [2, 14] and MC3R/MC4R-mediated signaling [21] are leptin-independent.

Ghrelin is the only circulating stomach-derived peptide and it functionally antagonizes leptin [23]. Ghrelin predominantly inhibits HPO axis [23‐25] through following 3 approaches: (i) Ghrelin directly inhibits Kiss1^AVPV [24] and GnRH neurons [25] in female rats. (ii) Ghrelin promotes the release of CRH in female rhesus monkeys, so it can indirectly repress GnRH neurons [23, 26]. (iii) Ghrelin stimulates NPY neurons and subsequently inhibits POMC neurons in rodents [27]. Although ghrelin primarily suppresses gonadotropin secretion in female animals and women [28], it benefits basal luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion in female rats [25]. Ghrelin in mouse placenta negatively modulates early embryonic development [23].

The anorexigenic insulin is found to activate HPO axis. Although in vitro mice study discovered that insulin can directly modulate GnRH neurons, in vivo studies of adult ewes and mice gave an opposite evidence [29, 30]. Indeed, insulin activates Kiss1^ARC in mice via functional insulin receptors [31]. Also, in laboratory animals, insulin excites ARC POMC neurons and suppresses NPY/AgRP neurons [30]. In addition, insulin in mice directly stimulates gonadotropes to enhance the LH mRNA expression [32].

The hepatic insulin-like growth factor-1 (IGF-1) enables to activate HPO axis. Firstly, both intracerebroventricular-infused and peripherally injected IGF-1 in the prepubertal female rodents can directly activates Kiss1^AVPV and GnRH neurons, thus leading to a precocious puberty [29]. Secondly, experiment in female rats demonstrated that low circulating IGF-1 caused by CR inhibits the pituitary gonadotropes, therefore represses the secretion of LH, FSH and thus estrogen [33]. Thirdly, IGF-1 signaling in ovine induces the activation of primordial follicles [34]. In addition, IGF-1 in mammalian ovary stimulates steroidogenesis, either alone or in synergy with gonadotropins [35].

The maintenance of follicle pool can reduce fertility and prevent premature ovarian failure [7, 57]. Compared with AL control group, CR in adult female rodents significantly increased the number of primordial follicles (PMFs). This finding indicates that CR reduces the rate of PMF activation, thus inhibiting the transition from primordial follicle to primary follicle [7, 42, 55‐59]. Secondly, the number of secondary follicles, antral follicles and corpus luteum were dramatically lower in CR-fed rodents. This observation suggests that CR suppresses the ovarian follicle development at different stages, follicle maturation and ovulation [39, 51, 53, 54]. CR inhibits follicle atresia because CR-fed mice and rats had the significantly low amount of atretic follicles [7, 42, 56, 57]. Also, CR inhibits the total follicle loss as the dramatically increased number of total surviving follicles was seen in CR-fed rodents [57‐59]. Although low fertility is observed in CR-fed mice, the capacity of fertility is augmented. Therefore, the fertility rebounds once AL-feeding is resumed [7]. It is noticed that CR also can augment the follicle pool and elongate the ovarian lifespan in adult female rats treated with chemotherapy [4]. SIRT1720, which partially mimics CR, achieves the similar effect in high-fat diet-induced obesity [58].

Figure 3a shows the mechanism that CR increases ovarian follicle pool. Initially it is found that CR enhances SIRT1, FOXO3a, NRF1 and SIRT6 gene expression in rodent ovary. More specifically, SIRT1, FOXO3a and SIRT6 are predominantly expressed in the oocytes and hardly expressed in the granulosa cells. Due to SIRT1-FOXO3a-NRF1 complex formed on the SIRT6 promoter can upregulate SIRT6 expression, activation of SIRT1/FOXO3a/NRF1-SIRT6 signaling is one of the avenues which CR hinders the transition from PMFs to primary follicles [57‐59, 62, 63]. SIRT1 upregulation by CR is important because it can also downregulate both p53 [53, 54] and mTOR complex 1 (mTORC1) [42, 58, 63] gene expression in rodent ovary. The evidence that low p53 inhibits follicle atresia is support by following studies: (i) p53 protein in rats is expressed in the apoptotic granulosa cells of atretic follicles [64]. (ii) Reduced p53 level in rat ovary is related to a significant decrease in the amount of apoptotic granulosa cells as well as atretic follicles [65]. (iii) p53 in mice is implicated in the regulation and selection of oocytes at checkpoints, such that oocytes that would otherwise be lost may persist when p53 is absent or reduced [66]. Recent studies from rodent models discovered that SIRT1 suppresses mTORC1-p70S6 kinase (S6K1)-ribosomal protein S6 (rpS6) signaling, thus preserving PMFs in quiescent state [42, 58, 63]. The most critical intra-oocyte signaling that controls PMF activation is the PI3K-Akt signaling. CR in female mice inhibits PI3K-Akt signaling and subsequently represses FOXO3a phosphorylation. The non-phosphorylated FOXO3a proteins are remained in oocyte nucleus, culminating in sustaining quiescent PMFs and thus maintaining ovarian follicle pool [55, 67]. Interestingly, it is found that CR preserving PMF pool is associated with low IGF-1 in rat ovary [33], and IGF-1 indeed activates PMFs via PI3K-Akt pathway in sheep ovary [34]. Therefore, CR may preserve PMF pool of rats by inhibiting IGF-1-PI3K-Akt signaling. It is also summarized that this signaling can upregulate mTORC1 expression [68]. In addition, CR overexpressing IGF-1 receptors (IGF-1Rs) may mediate the inhibition of follicle atresia because IGF-1Rs enable to antagonize cell apoptosis [33].

Two experiments in adult female mice give compelling evidence that CR enables to overcome the aging-related deterioration of egg quality: (i) The fecundity and postnatal offspring survival rate were remarkably increased in CR-then-AL fed mice [7]. (ii) The aging-related increases in aneuploidy, chromosomal misalignment on the metaphase plate, meiotic spindle abnormalities, mitochondrial aggregation and decreased ATP level, which were occurred in oocyte of AL-fed mice, were not exhibited in age-matched CR mice [61]. Therefore, good egg quality maintained by CR has a beneficial effect on oocyte meiotic maturation and fertilization, pre-implantation embryonic development, pregnancy success rate and embryo quality [60, 61].

The mechanism of CR keeping good egg quality is also shown in Fig. 3b. CR in adult mice upregulates mitochondrial SIRT3 in oocyte, and SIRT3 protect oocytes from the synthesis of mitochondrial reactive oxygen species (ROS). Therefore, high SIRT3 attenuates oxidative stress which declines oocyte quality with age [61, 62]. Also, CR in adult mice dramatically improves meiotic spindle assembly and maintenance, so it prevents oocyte aneuploid and chromosomal misalignment. In addition, CR enables to prevent the occurrence of aging-related mitochondrial dysfunction because it can appropriately arrange mitochondria in oocytes [61]. Although CR upregulates PGC-1α expression [60], Selesniemi et al. [61] discovered that loss of PGC-1α can reproduce the positive effect of CR on egg quality in aging female mice.

It is generally known that rodent models do not have true menses like humans. In humans, high size of PMF pool and slow rate of oocyte depletion are essential determinants of delayed menopause onset [69, 70]. As CR increases the number of PMFs and suppresses follicle development of rodents, it may have similar effect on human follicles. Therefore, CR seems to delay menopause onset and prolong reproductive lifespan of humans. However, the study of women who were exposed to Dutch famine discovered that CR decreases age at natural menopause, especially when occurring in early life [71]. The reason of this phenomenon is unknown. Another experiment showed an enigmatic discovery that the improvement of fecundity was observed in rabbits with CR alone [72]. Therefore, further studies are needed to explain these confusing findings.