Food restriction during pregnancy and female offspring fertility: adverse effects of reprogrammed reproductive lifespan

Maternal nutritional status during gestation is a key determinant for the health and physiology of the offspring at adulthood, and that influence is mainly established during early development, i.e. well before birth [1‐5].

Many adulthood diseases can be linked to the environment within which the embryo has developed, including abnormal nutritional, environmental, and hormonal insults that may have changed the developmental trajectory of the fetus [1, 2]. According to this hypothesis, the origins of common diseases may be due to the environment that the fetus directly senses via the mother [2, 5‐8]. In particular, maternal food restriction (MFR) has been associated with coronary heart disease and increased arterial blood pressure [2], reduced nephron endowment and increased renal morbidity in adulthood [9], and may affect physical growth and neurobehavior in newborns [10]. Malnutrition during gestation has been associated with hepatic steatosis, type 2 diabetes, and obesity during adulthood [5, 11‐15]. During late gestation, MFR is associated with metabolic signaling dysfunction in the liver, and predisposes the offspring to insulin resistance [5].

Oocyte quality is a critical determinant for the developmental trajectory of the fetus [16, 17]. Many forms of female reproductive disruptions have been linked to the prenatal environment, and it is likely related to early oocyte formation, which is vulnerable to numerous environmental effects [4]. In comparison with girls who were born appropriate for the gestational age, girls born small for the gestational age have a reduced reproductive lifespan, indicated by a decrease in ovarian size that is associated with low ovulation rates [18, 19], advanced menarche, and early menopause [20‐22]. Polycystic ovarian syndrome (PCOS), one of the most common female endocrine disorders [23], has been suggested to arise through a gene–environment interaction, probably in the developmental milieu within which female gametes are formed [24]. Although maternal malnutrition is a major factor that adversely affects fetal growth and is associated with to lifelong consequences, relatively few studies have investigated the effects of MFR on the reproductive outcomes of offspring [4, 25, 26]. In a large epidemiological study women born to mothers exposed to famine were more reproductively successful compared to controls [27]. Moreover, malnutrition during pregnancy alters reproduction in sheep by inducing poor oocyte quality, may cause reproductive disruptions in rats mainly by an early vaginal opening, and induce a decrease in the primordial and antral follicle number [28‐30]. During the first trimester of pregnancy in cattle, MFR leads to a reduced ovarian reserve in adulthood, as observed by the increased follicle stimulating hormone (FSH) levels [31].

At the moment, the available data regarding the relationship between the MFR and the female reproduction in the offspring have many limitations and gaps concerning the potential underlying mechanisms and almost all have evaluated the effects of maternal caloric restriction on the first generation only. Based on these considerations, the aim of the present experimental study was to investigate the impact of MFR in rats on ovarian architecture and function in first and second generations of female offspring.

The fertile reproductive lifespan of female mammals is mainly linked to the initial ovarian reserve of primordial follicles that reaches its maximum level around the time of birth, and is gradually depleted during reproductive life [40‐42]. At prepubertal age (week 4), we found that the number of primordial follicles was significantly decreased in FR1 and FR2 rats, suggesting that MFR affects the ovarian reserve of primordial follicles during early fetal life. The significant decrease in the number of primordial follicles at week 4 was associated with an increase in the number of primary follicles among the different studied groups suggesting that MFR might cause an early menarche by inducing early folliculogenesis. This hypothesis seems consistent with previous studies [26, 43].

At week 8, the significant decrease in ovarian weight related to the total number of follicles in both first and second generation offspring after MFR is mainly caused by the significant decrease in primordial follicles in the ovarian reserve compared to controls. This could suggest an early menopause in FR2, which is less likely in FR1 animals. In fact, previous studies reported that MFR is associated with early menarche and menopause [26, 43‐45]. To this regard, it is possible to hypothesize that MFR provided first and second generation offspring with a phenotype that is better suited for the lack of food. That new phenotype is consistent with the trade-off theory, i.e. an increase in fertility and a decrease in reproductive lifespan may lead to an increase in the chances of an organism to reproduce successfully [46]. In fact, fetal growth restriction can be considered as a part of the life history strategy for FR1 and FR2 females that were in utero when their mothers underwent food restriction. Since prenatal undernutrition leads to reduced longevity in mice [47], these females may anticipate a shorter life because of a higher risk of extrinsic mortality. It is possible to hypothesize that they may have to adjust their reproductive aptitude by changing the intensity and duration of their lifespan, the timing of the stages of folliculogenesis, as well as the age at which they should reach reproductive maturity. Due to the lack of food sensed through nutritional or endocrine signaling during fetal life [48], and to ensure reproductive success before death, these females have probably programmed their reproductive lifespan to be very intensive but relatively limited in time, which is consistent with population regulation in the theory of life history [49]. Thus, when they reach prepubertal age, they may upregulate the expression of genes involved in steroidogenesis, which in turn induces folliculogenesis for a greater number of primordial follicles while concomitantly explaining the significant higher number of in primary follicles in 4-week-old FR1 and FR2 females vs. control. The relatively large number of induced follicles undergoing folliculogenesis at one time makes follicles adjacent to each other and, consequently, highly increases the probability that they will merge to form MOFs [34]. This may have decreased the number of growing oocytes (secondary and antral follicles), and may explain the lower total number of follicles, and the higher number of MOFs in 4-week-old FR1 females (vs. controls). This strategy is also associated with a faster decline in ovarian function with aging, that is clearly supported by the significant decrease in the number of primordial follicles in the ovarian reserve, and also by the significant decrease in the total number of follicles in both FR1 and FR2 females at an early age (8-week), that corresponds in normal females to a high reproductive performance.

Of note, a higher number of MOFs in ovaries from 4-week-old first and second generation females was observed. Many follicles at these stages may have fused to form MOFs, which explains their significant higher number in FR1 females at four weeks, while this is less likely in FR2 females. While the mechanism of MOFs formation during nest breakdown has been described [33, 50], this is the second study that clearly confirms a new mechanism for the generation of MOFs through the fusion of follicles in the mammalian ovary, and their incidence increased sharply at prepubertal age [34]. Inversely, we found that the number of secondary and antral follicles was significantly lower in FR1 and FR2 at prepubertal age when compared to controls. Previous findings have reported that most cases of MOFs represent a fusion between secondary follicles, or a fusion between one secondary and one large antral follicle [34]. Furthermore, the number of secondary/antral follicles observed in 8-week-old females was higher than in controls when the number of MOFs was lower. This result is also consistent with the finding of Perez-Sanz and co-workers that showed that the number of MOFs declines significantly in female mice when they become sexually mature [33].

The presence of MNOFs is exceptional and represents a challenge for future studies. Two different mechanisms could explain the origin of this phenomenon: the nest breakdown–follicle assembly, and the fusion of more than one oocyte within the same multi-oocyte follicle. Based on our findings, the second mechanism appears the most probable since it is suggested that MOFs are most likely generated by the assembly of follicles [34] rather than being produced early on during the formation of the ovary. The presence of more than one oocyte in direct contact within the same follicle highly increases the possibility of their fusion, and leads to the formation of MNOFs. In fact, any cell brought into contact with another cell and given the right conditions (such as sufficient amounts of fusogen proteins, simultaneously present on each of the two cell surfaces) will fuse with the second cell, even when the latter is foreign [51‐53]. This fusion can be beneficial mainly during embryonic development, and for cell-based therapies, and represents a well-known process during reproduction when gametes (spermatozoa and oocytes) unite during fertilization to form the zygote. It has also been described in muscle cells, macrophages and nerve cells [54‐58]. When cell fusion is blocked during embryonic development, defects in organogenesis, embryonic lethality, and postembryonic defects can increase [53, 59] suggesting that fused cells are hybrid cells or chimera that function efficiently during embryonic development, driving correct organ formation [54]. In Caenorhabditis elegans, particularly in the proliferative zone of germ cells, two or more crescent shaped nuclei have been observed [60]. MNOFs have also been described during the initial stages of oogenesis in some amphibian species. For example, in Ascaphus truei, oogenesis involves eight nuclei [61], whereas frog oocytes with two nuclei have been described in Leiopelma hochstetteri [62]. The most evident example of the necessity for multi-nucleated cells is the syncytiotrophoblast, which is formed when embryonic cytotrophoblast cells fuse with the maternal endometrial epithelium [63, 64]. These cells represent the most important cell type in the placenta, and there is a strong correlation between a successful pregnancy and healthy syncytiotrophoblasts, likely due to their multi-nuclear state [64].

Current data suggest that MFR influences ovarian histopathology and, in turn, the reproductive health of first and second generation female offspring during fetal development, and they have been probably programmed to have an early menarche by inducing early folliculogenesis, and an early decline in ovarian function thereby decreasing the reproductive lifespan, and leading to an early menopause.