The mTOR pathway in reproduction: from gonadal function to developmental coordination

in Reproduction

Correspondence should be addressed to J Ramalho-Santos; Email: jramalho@uc.pt

Reproduction depends on many factors, from gamete quality to placenta formation, to fetal development. The mTOR pathway is emerging as a major player that integrates several cellular processes in response to a variety of environmental cues that are relevant in many aspects of reproduction. This review provides a general overview, summarizing the involvement of the two mTOR complexes (mTORC1 and mTORC2) in integrating signaling pathways, sensing environmental status, and managing physiological processes inherent to successful reproductive outcomes and pluripotent stem cell function. As a well-known governor of multiple cellular functions, it is not surprising that mTOR has a key regulatory role in determining cell quiescence or differentiation. In the gonads mTOR helps maintain spermatogonial stem cell and follicle identity and tightly regulates differentiation in both systems to ensure proper gamete production. The mTOR pathway is also known to prevent premature follicle exhaustion, while also controlling the blood–testis barrier in the male gonad. In stem cells mTOR again seems to have a role in controlling both pluripotency and differentiation, mirrored by its in vivo roles in the embryo, notably in regulating diapause. Finally, although there are clearly more complex systems intertwined in placental function, mTOR seems to serve as an early checkpoint for development progression and successful implantation.

Abstract

Reproduction depends on many factors, from gamete quality to placenta formation, to fetal development. The mTOR pathway is emerging as a major player that integrates several cellular processes in response to a variety of environmental cues that are relevant in many aspects of reproduction. This review provides a general overview, summarizing the involvement of the two mTOR complexes (mTORC1 and mTORC2) in integrating signaling pathways, sensing environmental status, and managing physiological processes inherent to successful reproductive outcomes and pluripotent stem cell function. As a well-known governor of multiple cellular functions, it is not surprising that mTOR has a key regulatory role in determining cell quiescence or differentiation. In the gonads mTOR helps maintain spermatogonial stem cell and follicle identity and tightly regulates differentiation in both systems to ensure proper gamete production. The mTOR pathway is also known to prevent premature follicle exhaustion, while also controlling the blood–testis barrier in the male gonad. In stem cells mTOR again seems to have a role in controlling both pluripotency and differentiation, mirrored by its in vivo roles in the embryo, notably in regulating diapause. Finally, although there are clearly more complex systems intertwined in placental function, mTOR seems to serve as an early checkpoint for development progression and successful implantation.

Introduction

Gamete production involves a series of complex meiotic processes, which occur in parallel with extensive morphological and physiological changes. Following fertilization, the one-cell zygote undergoes mitotic divisions culminating in blastocyst formation, accompanied by constant alterations in metabolism (Gardner & Harvey 2015), signaling pathways, chromatin arrangement and cell fate decisions (Bedzhov et al. 2014, Weinberger et al. 2016). In the blastocyst, the two first lineages are already segregated into the inner cell mass (ICM) and the trophectoderm (TE). The former will contribute to the embryonic tissues, and the latter, upon implantation, will contribute to the placenta that dynamically supports further development. In all these processes there is a constant need for tightly controlled cell divisions and precise environmental sensing, to ensure the quality of development.

The TOR protein was discovered in 1991 in the budding yeast, Saccharomyces cerevisiae, while uncovering the mechanism of action of rapamycin (Heitman et al. 1991), an efficient antiproliferative drug. A few years later, the mammalian analog of TOR was isolated and named mammalian Target Of Rapamycin (mTOR; Brown et al. 1994, Sabatini et al. 1994) or mechanistic Target Of Rapamycin. As a major player in cell signaling, mTOR coordinates several fundamental processes such as cytoskeletal organization (Jacinto et al. 2004, Sarbassov et al. 2004), cell survival (Stambolic et al. 1998, Hirose et al. 2014), autophagy (Nicklin et al. 2009, Koren et al. 2010), lipid synthesis (Li et al. 2011, Peterson et al. 2011), protein synthesis (Ma & Blenis 2009, Laplante & Sabatini 2012) and the cell cycle (Fingar et al. 2004, Gao et al. 2004, Chatterjee et al. 2015), among others (Fig. 1). More importantly, outcomes of these distinct pathways depend on key environmental cues, including the presence/absence of growth factors (Laplante & Sabatini 2012), oxygen/hypoxia (Brugarolas et al. 2004, DeYoung et al. 2008), energy status (Inoki et al. 2003a), nutrients (Kim et al. 2002, 2008, Smith et al. 2005) and other stressors (Fig. 1). This complex integration of diverse stimuli has an important role in cell homeostasis, proliferation and differentiation. Consequently, mTOR signaling impairment can lead to severe physiological dysfunction at many levels. Considering the global nature of this signaling pathway, this review aims to integrate the role of mTOR in reproduction, providing a brief overview of its importance, from gonads and gametes, to embryos, stem cells and the placenta.

Figure 1
Figure 1

The integrative role of the mTOR pathway on cellular homeostasis. mTORC1 and mTORC2 assemble differently, relying on particular allies to respond to distinct stimuli and accordingly regulate specific cellular outcomes.

Citation: Reproduction 159, 4; 10.1530/REP-19-0057

mTOR: more complex than just a protein

mTOR is a highly conserved protein that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family. The ability of this serine/threonine kinase to sense different cues and interact with particular cellular targets is dependent on its ability to interact with other proteins, forming two multimeric complexes – mTORC1 and mTORC2. RAPTOR is mandatory for the assembly of the mTORC1 complex (Kim et al. 2002, Sancak et al. 2007). On the other hand, the assembly of the mTORC2 complex relies on mSIN1 and RICTOR(Sarbassov et al. 2004) and on RICTOR association with PROTOR 1/2 (Pearce et al. 2007, 2011). Regardless of being assembled with the partners that characterize mTORC1 or mTORC2, the mTOR protein is capable of binding to mLST8, DEPTORr and the Tti1/Tel2 complex (Laplante & Sabatini 2012).

mTORC1 is sensitive to growth factors, hypoxia, energy status (via AMPK activity) and nutrients. Consequently, mTORC1 activity promotes translation and protein synthesis, mainly through two mTORC1 substrates: ribosomal protein S6 kinases (S6Ks) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1; Ma & Blenis 2009, Laplante & Sabatini 2012). Furthermore, mTORC1 promotes lipid biosynthesis by modulating the activity of SREBP1/2 (Li et al. 2011, Peterson et al. 2011) and PPARG (Kim & Chen 2004, Zhang et al. 2009). On the other hand, autophagy is negatively regulated by mTORC1 via several mechanisms (Laplante & Sabatini 2012), namely, by activating the autophagy suppressor DAP1 (Koren et al. 2010), and also by regulating TFEB and preventing lysosome biogenesis (Settembre et al. 2012). Additionally, mTORC1 regulates glucose metabolism and mitochondrial function by modulating HIF1A and PGC1A (Brugarolas et al. 2003, Cunningham et al. 2007, Düvel et al. 2010).

Growth factors activate PI3K- or Ras-dependent signaling pathways that, in turn, can regulate mTORC1, leading to TSC1 and TSC2 complex phosphorylation, inactivating its GAP activity (Inoki et al. 2002, Manning et al. 2002, Tee et al. 2003). This triggers the Rheb GTPase, which in its GTP-bound state (Inoki et al. 2003b) directly activates mTORC1, via phosphorylation (Inoki et al. 2002, Manning et al. 2002, Potter et al. 2002, Long et al. 2005, Sancak et al. 2007). On the other hand, AMPK is sensitive to the AMP:ATP ratio, making AMPK a major cellular energy sensor (Shackelford & Shaw 2009, Hardie et al. 2012). Accordingly, when this ratio increases, AMPK also activates mTORC1, and other stressors such as DNA damage may also regulate mTORC1 via the regulation of REDD1 (Brugarolas et al. 2004, DeYoung et al. 2008). Moreover, amino acid availability was shown to regulate mTORC1 similar to its inhibitor rapamycin (Hara et al. 1998). Amino acid withdrawal leads to dephosphorylation of direct mTORC1 targets S6K1 and 4EBP1, inhibiting mRNA translation (Hara et al. 1998), an effect that is reversed upon amino acid reconstitution in cell culture media. Although the exact mechanism of how amino acid availability activates mTORC1 is still unclear, pathways involving Rag GTPases (Kim et al. 2008) or biogenic polyamines have been proposed (Zeng et al. 2013, Ray et al. 2015). However, it should be noted that not all amino acids display the same efficiency in stimulating mTORC1 (Hara et al. 1998, Long et al. 2005). Finally, impairment in the transport or the lack of amino acids also affects cell proliferation in an mTOR -dependent manner (Smith & Proud 2008, Nicklin et al. 2009, González et al. 2012) (Fig. 2).

Figure 2
Figure 2

Upstream signals that affect mTOR regulation. Different stimuli, namely, growth factors and hormones, insulin, energy stress (AMP/ATP ratio), hypoxia and nutrient availability, are known to modulate mTORC1 and mTORC2 differently, acting through different signaling cascades.

Citation: Reproduction 159, 4; 10.1530/REP-19-0057

In contrast to mTORC1, mTORC2 is less well understood, although it is known to be activated in a PI3K-dependent fashion and thus is also responsive to growth factors. Furthermore, its main targets are AKT, SGK1 and PKCA, thus regulating cell survival and growth (Stambolic et al. 1998, Laplante & Sabatini 2012), ion transport (Potter et al. 2002, García-Martínez & Alessi 2008) and cell shape through cytoskeletal reorganization (Jacinto et al. 2004, Sarbassov et al. 2004). mTORC2 has also an indirect role in metabolism, not only in terms of mitochondrial physiology (Betz et al. 2013) but also by being able to control glucose metabolism in AKT-dependent (Hagiwara et al. 2012, Roberts et al. 2013, Masui et al. 2014) and FOXO-dependent (Hagiwara et al. 2012, Masui et al. 2013) manners. Finally, mTORC2 is also able to modulate the hexosamine biosynthetic pathway (Moloughney et al. 2016), thus affecting protein and lipid glycosylation, as well as glutamine metabolism (Boehmer et al. 2003, Moloughney et al. 2016), both involved in anabolic processes.

It should be noted that both mTOR complexes can intrinsically regulate each other via a feedback mechanism that has not been completely characterized. Of the known mTORC2 targets, AKT is the most intensively studied, followed by mTORC2-mediated serine 473 phosphorylation. Further, AKT targets TSC2, resulting in mTORC1 activation (Laplante & Sabatini 2012). In contrast, mTORC1 can limit mTORC2 activity by stimulating S6K1, that can, in turn, inactivate mTORC2 by direct phosphorylation of mSIN1 (Liu et al. 2013) and RICTOR (Julien et al. 2010, Fig. 2).

(Sper)mTOR: mTOR activity in testicular function

Infertility is classically defined as the inability to conceive after frequent and unprotected sexual intercourse for over a year (WHO 2010). It affects about 10% of couples worldwide (Mascarenhas et al. 2012), and approximately 50% of all cases are attributed to the male partner (WHO 2010, Miyamoto et al. 2012). During spermatogenesis, spermatogonia undergo a few mitotic divisions and then differentiate into primary spermatocytes that undergo meiotic divisions to yield haploid spermatids, which differentiate to mature spermatozoa (Holstein et al. 2003, Cheng & Mruk 2012). Unsurprisingly, this process is highly controlled by a wide range of signaling pathways, including mTOR.

The first evidence of a possible impact of mTOR on male fertility was related to the clinical report of an immune-compromised patient due to sirolimus (rapamycin) treatment, showing a dramatic decrease in sperm quality, namely, sperm count (oligospermia), percentage of normal-shaped sperm (teratospermia) and sperm motility (asthenospermia). Importantly, these negative effects of rapamycin treatment were completely reversible (Bererhi et al. 2003). In line with these effects on sperm quality, several case reports subsequently showed that rapamycin treatment led to decreased testosterone levels and increased levels of FSH and LH, reinforcing the hypothesis that this mTOR inhibitor could be having an adverse effect on testicular function (Fritsche et al. 2004, Lee et al. 2005). These patients also had severe oligoasthenospermia and in some cases complete azoospermia (complete lack of sperm in the ejaculate), which, again, was reversible after the treatment ended (Deutsch et al. 2007, Skrzypek & Krause 2007, Zuber et al. 2008, Boobes et al. 2010).

In agreement with these reports, rats exposed to rapamycin presented lower testicular weight and a decreased seminiferous tubule area. Additionally, in the testes of these animals, only spermatogonia were found but not spermatocytes or spermatids, suggesting that mTOR inhibition blocked spermatogenesis at the spermatogonial level (Rovira et al. 2012). Rapamycin-treated rats presented not only smaller and vacuolated seminiferous tubules but also decreased sperm production and blocked spermatogonial cell cycle progression (with increased percentage of spermatogonial cells in G1), suggesting inhibited spermatogonia proliferation that could explain the impaired sperm production, although entry into meiosis might also be affected (Xu et al. 2016).

Indeed, in mice, rapamycin decreased the expression of meiotic markers, suggesting that mTOR inhibition maintains spermatogonia in the undifferentiated state (Busada et al. 2015, Sahin et al. 2018). More importantly, a spermatogonial-specific over-activation of the mTORC1 complex led to testicular development defects, sperm count reduction and increased germ cell apoptosis. In addition, mTORC1 activation induced spermatogonial differentiation, which, combined with increased germ cell apoptosis, resulted in the depletion of the germ cell pool (Hobbs et al. 2010, Zhou et al. 2015, Wang et al. 2016). It is therefore unsurprising that a conditional germ cell mTOR knockout (cKO) resulted in smaller testes with abundant spermatogonia and no apparent spermatocytes, confirming impairment in germ cell differentiation. Interestingly, a subset of undifferentiated spermatogonia in the mTOR cKO mouse could still self-renew, suggesting that mTOR is dispensable for the maintenance of undifferentiated spermatogonia but necessary for differentiation and spermatogenesis (Serra et al. 2017). In agreement with this observation, differentiation-prone spermatogonia have elevated mTORC1 activity, when compared to their undifferentiated stem cell counterparts, suggesting that mTORC1 may negatively regulate spermatogonial stem cell self-renewal (Hobbs et al. 2015).

It is clear that mTOR is highly involved in spermatogenesis; however, the exact mechanism by which it impacts the spermatogonial pool is still under discussion, as some studies have shown that mTOR modulation impacts spermatogonial proliferation and apoptosis, while others suggest that it is not a major player in self-renewal but is crucial for spermatogonial differentiation. Another important fact to consider is that the various methodological approaches used in different papers resulted in different rates of modulation for each of the mTOR complexes, leading to distinct outcomes. Nonetheless, a germ cell-specific Rictor cKO mouse model presented significant spermatogenesis impairment, with some tubules exhibiting the loss of germ cell hierarchy including elongated spermatids at the basal membrane, while other tubules were germ cell-depleted, probably due to an increase in preleptotene and leptotene spermatocyte apoptosis. Importantly, the loss of Rictor caused an upregulation of cell adhesion and migration-associated genes in Sertoli cells, suggesting that mTOR has a role in not only the regulation of spermatogonial proliferation but also the integrity of the blood–testis barrier (BTB; Bai et al. 2018).

The BTB is formed by adjacent Sertoli cells at the basement membrane of the seminiferous tubules with intercellular spaces sealed by tight junctions (TJs) at the apical region, while ectoplasmic specialization (ES) junctions, namely, adherens junctions (AJs), provide links not only between Sertoli cells but also between Sertoli and germ cells (Mok et al. 2013a). In addition, the basal region of the BTB depends on desmosomes and gap junctions (GJs) for its cohesion (Cheng & Mruk 2012, Mok et al. 2013a).

The BTB undergoes cyclic reconstruction during spermatogenesis, to allow the transit of preleptotene spermatocytes into the adluminal region during stages VIII to XI of the testicular epithelial cycle (Mok et al. 2013a,b). The mTOR protein is highly expressed at the BTB, suggesting an important role in its modulation, and over-activation by conditional deletion of Tsc1 led to mislocalization of several proteins involved in testicular junctional complexes (Tanwar et al. 2012). Furthermore, phosphorylated pS6, a downstream target of mTORC1, is expressed specifically at late stages VII and XI of the spermatogenic cycle and is co-localized with several proteins of the BTB, namely, ZO-1 (a TJ-associated protein), N-cadherin (an ES protein) and F-actin, in a stage-specific manner (Mok et al. 2012, 2015). Importantly, the inhibition of mTORC1 by rapamycin and S6 Knockdown (cKD), not only strengthened the BTB by increasing the expression of TJ-associated proteins but also increased the proportion of F-actin bundle alignments (Mok et al. 2012). This suggests that the activation of the mTORC1 complex is necessary to weaken the BTB and allow the transit of preleptotene spermatocytes to the adluminal region of the seminiferous tubules (Mok et al. 2012, 2015).

On the other hand, the mTORC2 complex protein, RICTOR is partially co-localized with TJ proteins, and its expression shifts with the seminiferous cycle stage. Although both RICTOR and mTOR are highly expressed at stages V to VI, at stage VII, RICTOR expression starts to decline until it is barely detectable at stages VIII to IX. To note, the high mTOR expression throughout these late stages supports the hypothesis that mTORC2 assembly is required in the first stages of the spermatogenic cycle to maintain the integrity of the BTB but is dispensable and downregulated during stages VIII and IX, when the barrier restructuring takes place (Mok et al. 2013b). Interestingly, when Rictor was conditionally knocked down in Sertoli cells, there was no downregulation of TJ proteins, but these proteins were mis-localized from the Sertoli–Sertoli interface. Moreover, F-actin bundles were disorganized, suggesting that mTORC2 assembly is important for the maintenance of a cohesive BTB (Mok et al. 2013b). In accordance, Rictor cKO mice had severe testicular defects, as they had no seminiferous lumen or elongated spermatids and, more importantly, presented a loss of germ cell hierarchy in the seminiferous epithelium (Dong et al. 2015) that could explain the resulting azoospermia. In agreement, Mok et al. (2013b) reported that Rictor ablation did not change the protein levels of occluding (TJ protein), N-cadherin or actin-related protein complex 2 (Arpc2) but altered their localization, weakening the BTB (Dong et al. 2015). Interestingly, mTOR cKO in Sertoli cells produced a similar disorganization of the seminiferous epithelium, which seemed to be due to a redistribution of the BTB gap junction protein 1 (GAP-1) (Boyer et al. 2016). Thus, the mTOR pathway is a major regulator of the BTB permeability, given that a timely activation of both mTORC1 and 2 is required to maintain a cohesive and flexible barrier.

In summary, the mTOR pathway is involved in spermatogenesis, by regulating spermatogonial germ cell proliferation and apoptosis and by tightly controlling BTB function. However, further research is still needed to evaluate the effect, if any, of mTOR on the differentiated functional sperm cell (Silva et al. 2019). It is intriguing to speculate that an effect of mTOR on male fertility may be in some way related to energy sensing and nutrient availability, which could condition male investment in gamete production, depending on environmental cues. Considering that after ejaculation sperm must travel through the female reproductive tract to fertilize the oocyte and that the mTOR pathway is regulated by hypoxia and extracellular pH, it would be interesting to assess how the female reproductive tract environment could modulate sperm function.

mTOR in female reproduction: keep it slow or speed it up

Following the migration and establishment of primordial germ cells (PGCs) in the developing ovary, a process named primordial follicle assembly takes place, in which PGCs are surrounded by somatic cells (namely, squamous granulosa cells (GCs)). When squamous GCs assume a cuboid morphology, the primary follicle is formed. The GCs start to proliferate and recruit steroidogenic cells (theca cells), leading to the secondary follicle formation. The antral follicle is characterized by the appearance of a cavity filled with follicular fluid (antrum), which divides two populations of GCs: the more external mural GCs and the cumulus GCs. This follicle growth will give rise to the pre-ovulatory Graafian follicle. Female meiosis is initiated during fetal development, and the oocytes enclosed in primordial and primary follicles are arrested in diplotene of prophase I, also known as the germinal vesicle (GV) stage. After primary follicle activation, GV envelope breaks down (GVBD) and meiosis progresses until it is once again arrested at metaphase II. It is at this stage that oocytes are ovulated in most mammals, and meiosis is completed only if fertilization takes place.

The PI3K/AKT/mTOR pathway regulates events that involve follicle quiescence, activation, development, proper oocyte maturation and ovulation. Because this pathway responds to several growth factors and hormones, it is not surprising that it is sensitive to LH signaling in theca cells (Palaniappan & Menon 2010, 2012) and FSH signaling in GCs (Kayampilly & Menon 2007, Sirotkin et al. 2015). Several studies suggest that mTOR is a positive regulator of GC proliferation (Kayampilly & Menon 2007, Yaba et al. 2008, Yu et al. 2011); follicular survival, activation and development (Cheng et al. 2015, Zhang et al. 2017a, Li & Liu 2018); and translation of maternal transcripts (Chen et al. 2013). Furthermore, its deregulation is associated with follicular atresia and ovarian function impairment (Choi et al. 2014, Chen et al. 2015a). For those outcomes, communication between oocytes and GCs is of utmost importance. In oocytes the disruption of the negative regulators of mTORC1 TSC1 (Adhikari et al. 2010) or TSC2 (Adhikari et al. 2009) led to the over-activation of primordial follicles and infertility. This reinforces the notion that mTOR activity through oocyte–GC communication is crucial to maintain primary follicles in a quiescent state. On the other hand, Rictor cKO on oocytes showed that mTORC2 activity seems to be mostly associated with follicle survival in an oocyte-dependent fashion (Chen et al. 2015a). Moreover, the fact that mTOR cKO in primordial oocytes causes a shift in GCs morphology to an immature Sertoli cell-like phenotype, including the expression of genes characteristic of this testicular cell type (Guo et al. 2018), suggests that the communication between oocytes and the surrounding GCs is crucial to sustain cellular identity, although not much is known about how exactly mTOR is regulating this.

Given these results, mTOR has emerged as a putative target to ameliorate either fertility dysfunction related to premature follicle pool loss or exhaustion of the oocyte pool, and therefore in modulating aging-specific effects. Thus, several rodent models of premature ovarian failure (POF) and primary ovarian insufficiency (POI) conditions, characterized by premature follicle depletion, revealed that mTOR deregulation led to anomalous primary follicle activation (Adhikari et al. 2009, 2010, 2013, Sherman et al. 2014). Furthermore, rapamycin restricts the transition from primordial to developing follicles in rodent models, reducing follicular atresia and extending ovarian lifespan by downregulating mTOR activity and increasing SIRT activity, similar to caloric restriction (Luo et al. 2013, Zhang et al. 2013). However, although prolonged rapamycin exposure delays follicle pool exhaustion, it has detrimental consequences on murine ovarian function and thus on the reproductive cycle, which may be overcome by transient treatments of rapamycin (Dou et al. 2017), although possible persistent side effects require further attention.

Furthermore, in terms of putative effects on gametogenesis, it is important to mention that mTOR disruption has been reported to cause errors during meiotic and mitotic chromosome disjunction (Lee et al. 2012, Yu et al. 2012, Dou et al. 2017). In interphase GCs mTOR has a predominant cytoplasmic distribution (as is the case for its phosphorylated form) but is also found in the nuclei. In contrast, during prophase, its localization shifts to the vicinity of chromosomes and becomes more intense in the spindle during metaphase and cytokinesis (Kogasaka et al. 2013). Interestingly, RAPTOR, part of mTORC1, co-localizes with mTOR in all these phases of the mitotic process (Yu et al. 2011, Kogasaka et al. 2013), while mTORC2-specific RICTOR seems to be dissociated from mTOR throughout mitosis (Kogasaka et al. 2013). This indicates that mTORC1 and mTORC2 are differentially active, not only during mitosis in GCs but also during meiosis in oocytes. In mature metaphase II oocytes mTOR localization at the spindle (Lee et al. 2012, Kogasaka et al. 2013) may be related to its repressive effect on autophagy, given that low levels of autophagy are observed in oocytes, increasing dramatically upon fertilization (Tsukamoto et al. 2008). Despite all the correlative evidence concerning mTOR localization during meiosis, it is still unknown if the effects are directly related to mTOR function in situ or are a consequence of its activity in previous phases of oocyte development.

mTOR and early embryo development

After fertilization the totipotent zygote undergoes mitosis, and the 8- to 16-blastomere embryo experiences a process known as compaction (Ziomek & Johnson 1980, Fierro-González et al. 2013). Accordingly, an outer layer of polarized and non-polarized blastomeres is defined, surrounded by more internal cells containing non-polar blastomeres (Yamanaka et al. 2010, McDole et al. 2011). In the 32-blastomere-compacted morula, there is evidence that cells are already more committed to one of the two first embryo cell lineages: the outer cells will give rise to the trophectoderm (TE), and the inner cells to the pluripotent ICM (McDole et al. 2011, Goolam et al. 2016, Posfai et al. 2017). As development proceeds, pluripotency genes are gradually restricted to internal non-polarized cells via TEAD4 activity (Yagi et al. 2007, Nishioka et al. 2008). Thus, TE cells will be specified by Cdx2 (Nishioka et al. 2009, Schrode et al. 2013) and Gata3 (Ralston et al. 2010, Schrode et al. 2013) expression, whereas the ICM will be specified by the expression of Oct4, also known as Pou5f1 (Nichols et al. 1998, Nichols & Smith 2012), Nanog (Chambers et al. 2003, Mitsui et al. 2003) and Sox2 (Avilion et al. 2003).

Within the compacted morula a cavity full of fluid (the blastocoel) forms, giving rise to the blastocyst, in which TE cells are arranged side to side in a more external layer of the embryo, surrounding both the ICM and the blastocoel (Bedzhov et al. 2014). The transition from early to late blastocyst comprises the spatial segregation of the epiblast (EPI) composed of pluripotent stem cells (PSCs) – also known as embryonic stem cells (ESCs) – and the hypoblast composed of primitive endoderm (PrE) cells, allocating PrE cells between the EPI and blastocoel (Yamanaka et al. 2010, Leung & Zernicka-Goetz 2015). All the three cell types of the pre-implantation blastocyst have different developmental destinations. In the conceptus, the EPI cells will originate the fetus, while the PrE will form the yolk sac, and the TE cells will contribute to the placenta (Schrode et al. 2013, Leung & Zernicka-Goetz 2015).

In the mouse homozygous mutations for mTOR are lethal, and blastocysts fail to develop, displaying proliferation flaws of both embryonic and extraembryonic tissues (Murakami et al. 2004). Moreover, proliferation of both ICM and trophectoderm cells from these embryos was impaired in vitro, which was also observed in mutants lacking amino acid residues on the mTOR C terminus, critical for its kinase activity (Murakami et al. 2004).

To better understand the role of the mTOR pathway in both embryo development and in ESCs, studies have targeted the specific binding partners and upstream regulators of mTOR complexes. Thus, the lack of PI3K or Akt family members or subunits, both upstream regulators of mTOR activity, resulted in lethality or developmental defects (Peng et al. 2003, Tschopp et al. 2005, Yang et al. 2005, Foukas et al. 2006, Zhou et al. 2011, Yoshioka et al. 2012). Mutants of the direct regulators of mTORC1, Tsc1 (Kobayashi et al. 2001), Tsc2 (Rennebeck et al. 1998) and Rheb (Goorden et al. 2011) died approximately at the same developmental time window, with ectodermal and mesodermal lineage development defects. Interestingly, mutants of Raptor (mTORC1) become non-viable at the same developmental time as that of mTOR mutants, while Rictor (mTORC2) mutants die later due to fetal cardiovascular and nervous system abnormalities (Guertin et al. 2006, Shiota et al. 2006), indicating that each complex is involved in different developmental processes.

mTOR was also identified as a master regulator of embryonic diapause (ED – Box 1; Bulut-Karslioglu et al. 2016). In fact, culturing embryos in the presence of a pharmacological mTOR inhibitor arrested embryo development for 9–12 days at the blastocyst stage, an effect that was reversible upon the inhibitor removal. Moreover, this work was extended to PSCs, inducing a novel state of paused pluripotency in which cells presented a hypotranscriptome, proliferating less in the presence of the inhibitor. The pausing effects were reversible without any negative impact on pluripotency. Earlier the same year the myelocytomatosis oncogene (Myc) had already been suggested as a central player promoting ED, since transcriptional factor family members are under-expressed in paused embryos and KO or pharmacological inhibition of Myc members in mouse embryos, and mESCs also lead to a reversible diapause-like state in both pluripotent cells and embryos (Scognamiglio et al. 2016). However, this stage was sustained for only a few hours in culture, pinpointing that an upstream signal would be a best candidate to control developmental pausing, which is plausible given that Myc acts downstream of mTOR (Masui et al. 2013).

Box 1: Embryonic Diapause

Diapause is a very conserved phenomenon across nature, from plants to mammals (MacRae 2010), though it does not happen equally in all living systems. ED is achieved when embryo development is very slow or even temporarily arrested (Fenelon & Renfree 2018). Two forms of ED are currently known: obligatory or facultative. Obligatory diapause is species-specific and happens at a well-defined stage of development or in response to environmental stimuli such as photoperiod (Renfree & Fenelon 2017). It occurs in some bats, bears and marsupials (Fenelon et al. 2014). Facultative diapause is a consequence of adverse conditions such as lactation or metabolic stress (e.g. starvation), when further embryo development could be risky (Lopes et al. 2004). This seems to be a very sophisticated mechanism to ensure that proper conditions for development to occur are available, optimizing offspring survival.

Facultative ED is frequent in some mammals such as mice. The hormonal regulation during lactation makes the uterus a hostile environment for implantation (Renfree & Fenelon 2017); however because female mice can mate instantaneously after giving birth, ED is a strategic mechanism to guarantee the survival of the next generation without jeopardizing the current litter. Consequently, embryos develop until the blastocyst stage, and when there is contact with the uterine wall (apposition), they become dormant. During ED the rate of proliferation and growth becomes slower, accompanied by a decrease in DNA, RNA and protein synthesis. Furthermore, a hypometabolic state and an improvement of stress resistance are established (MacRae 2010, Fenelon et al. 2014). As soon as the proper conditions arise or upon estrogen administration, development is resumed and embryo implantation quickly takes place (Renfree & Fenelon 2017). Thus, communication between the embryo and the uterine tissue appears to be critical in regulating diapause (Nichols et al. 2001, Fenelon et al. 2014).

The role of ovarian hormones on endometrial receptivity is clear, given that embryos from ovariectomized mice cannot implant and that estrogen and progesterone are important for uterine receptivity (Chen et al. 2000). Furthermore, estrogen is also essential for uterine LIF expression, which, in turn, is fundamental for implantation (Stewart et al. 1992, Chen et al. 2000). Female LIF-deficient mice generate viable embryos that cannot implant unless they are transferred to a pseudo-pregnant recipient (Stewart et al. 1992), and diapause embryos lacking part of the LIF receptor heterodimer are unable of develop (Nichols et al. 2001) demonstrating the importance of LIF in this process.

Considering the high plasticity of the embryo, the mTOR pathway may be somewhat redundant, and compensatory mechanisms could assure viability until the embryo develops to the blastocyst stage, where mTOR activity is, in fact, crucial for further development. Considering that the KO of mTOR regulators and binding partners display different embryonic phenotypes, it is not clear if the role of mTOR at this level is solely dependent on its kinase activity, as suggested by Murakami et al. (2004), or if it regulates cellular processes during earlier embryonic events that may be required for further blastocyst development (e.g. epigenetic alterations).

mTOR and pluripotency regulation

Pluripotency is a transient state during embryo development, but different pluripotent states can be recapitulated in vitro with defined media formulations (Nichols & Smith 2012, De Los Angeles et al. 2015, Weinberger et al. 2016), allowing for the study of pluripotency regulation in vitro and comparisons to in vivo counterparts (Box 2). Changes in pluripotency and cell fate commitment involve not only metabolic and chromatin remodeling (Boroviak et al. 2014, Zylicz et al. 2015, Mathieu & Ruohola-Baker 2017) but also signaling pathways (Illich et al. 2016, Weinberger et al. 2016).

Box 2: Pluripotency Regulation

Naive mESCs resemble ICM cells and express Oct4, Sox2 and Nanog, which are core pluripotency establishment and maintenance genes, acting through positive-feedback loops that promote their own expression and suppressing the expression of genes that trigger differentiation (Young 2011).

Other genes such as Klf4, Klf2, Esrrb, Tfcp2l1, Tbx3 and Gbx2 are involved in self-renewal and naive pluripotency preservation (Tai & Ying 2013, Boroviak et al. 2014, Weinberger et al. 2016), and in some cases their expression is promoted by LIF signaling (Niwa et al. 2009, Tai & Ying 2013, Martello et al. 2013, Ye et al. 2013) and the activity of the GSK3B inhibitor CHIR (also known as CHIR99021) (Wray et al. 2011, Martello et al. 2012). Moreover, using PD03, a MAPK/Erk inhibitor also enhances naive state propagation avoiding Erk-mediated chromatin modulation to promote the transcription of lineage-commitment genes (Tee et al. 2014).

mESC culture conditions have been improving throughout the years with the development of a medium supplemented with LIF and the two specific inhibitors noted earlier. This 2i media is sufficient to maintain naive pluripotency and self-renewal. Even though LIF signaling is dispensable for naive cell maintenance, its presence is beneficial for proliferation (Ying et al. 2008). On the other hand, mEpiSCs downregulate the expression of many of the genes from the pluripotency circuitry controlling naive cells pluripotency, such as Gbx2, Tbx3, Esrrb, Rex1 (Weinberger et al. 2016), while maintaining the expression of Oct4 and Sox2 (Tesar et al. 2007, Guo et al. 2009). At the same time, primed cells heterogeneously upregulate the expression of lineage-commitment genes, such as T-brachyury and Fgf5 (Tesar et al. 2007, Guo et al. 2009). Naive and primed cells differ not only in their developmental origin. EpiSCs, as mentioned, can be derived from the post-implantation embryo and can also be obtained by the differentiation of mESC by culturing cells in the presence of Fgf2/Activin A (Guo et al. 2009, Weinberger et al. 2016). Interestingly, mEpiSCs can differentiate into cells of the three germ layers in embryonic bodies and teratoma assays but inefficiently contribute toward chimeras (Brons et al. 2007) when injected in the ICM of pre-implantation embryos, probably due to their developmental incompatibility with the ICM of the pre-implantation embryo.

Serum/LIF cultured PSCs represent a transient state between the naive and the primed state, since cells in this culture system are highly heterogeneous. Although the core pluripotency transcription factors are expressed, genes involved in naive cell self-renewal are heterogeneously expressed in serum/LIF cells (Chambers et al. 2007, Niwa et al. 2009), becoming more vulnerable to differentiation. Yet cells that downregulate these genes also seem to be able to re-express them (Chambers et al. 2007) and therefore are not completely committed.

Pluripotent mouse embryonic stem cells (mESC) were derived from the ICM of diapause blastocysts and cultured in vitro for the first time in 1981 (Evans & Kaufman 1981, Martin 1981). These cells share many features with the epiblast (EPI) cells of the pre-implantation embryo (Boroviak et al. 2014), and they were later tagged as “naive” PSCs. There is another pluripotent state, described as the “primed” state, involving mEpiSCs, which resemble the post-implantation EPI cells of the embryo from where they were derived (Brons et al. 2007, Tesar et al. 2007, Nichols & Smith 2012, Boroviak et al. 2014).

Stem cells can undergo an undefined number of divisions in culture, always maintaining the stem cell pool (self-renewal) without losing their ability to differentiate into specific lineages according to their potential (De Los Angeles et al. 2015). PSCs can differentiate into cells from the three germ layers, and this potential can be assessed using teratoma and chimera generation assays, which in theory should give rise to characteristic mesoderm, endoderm and ectoderm cell types (Bradley et al. 1984, Huang et al. 2012, De Los Angeles et al. 2015).

In vivo the mTOR pathway is known to be involved in suppressing mesendoderm commitment (McLean et al. 2007, Zhou et al. 2009, Yu et al. 2015, Jung et al. 2016). While mTORC1 disruption appears to trigger early differentiation, mTORC2 seems to be important during later stages, rather than in early lineage priming (Zhao et al. 2014, Lu et al. 2017, Zheng et al. 2017a,b). mTOR activity has also been reported to enhance ectoderm differentiation (Freund et al. 2008, Jung et al. 2016), which could possibly be a consequence of preventing cell differentiation toward other germ layers.

In cultured ESCs mTOR has been associated with self-renewal and pluripotency maintenance while preventing differentiation (Zhou et al. 2009, Ryu and Han 2011, Betschinger et al. 2013, Cho et al. 2014, Jung et al. 2016). In accordance, rapamycin, which targets mTORC1, can disrupt pluripotency by increasing differentiation rates in mesoderm/endoderm through a transient suppression of this complex (Zhou et al. 2009, Lu et al. 2017). However, this effect may not be solely mediated by mTORC1, given that prolonged exposure to rapamycin also disrupts mTORC2 (Sarbassov et al. 2006), raising the question of which mTOR complex plays the more crucial role in pluripotency. In contrast, other reports show that phosphorylated levels of S6K1 (a direct target of mTORC1) are reduced in hESCs when compared to differentiated cells (Easley et al. 2010), as are overall translation rates (Sampath et al. 2008, Easley et al. 2010). This may be closely associated with the balance between pluripotency and the translation of differentiation factors. Moreover, the mTOR-driven increase of S6K1 activity induces differentiation (Easley et al. 2010) and transient suppression of mTORC1 activity, with rapamycin enhancing the efficiency of pluripotency induction (Wang et al. 2013), indicating that mTORC1 inhibition favors both pluripotency maintenance and induction. Furthermore, the expression of DEPTOR, an intrinsically negative regulator of both mTORC1 and mTORC2, is increased in ESCs and downregulated during differentiation (Agrawal et al. 2014), modulating pluripotency by stabilizing Oct4, Nanog and Zfp42 expression.

mTOR activity has also been linked to glucose metabolism regulation downstream of LIN28, facilitating the transition from a naive to a primed pluripotency state by supporting high glycolysis flux and nucleotide metabolism (Zhang et al. 2016). Studies using other cell types revealed that mTORC1 is involved in the regulation of nucleotide synthesis (Düvel et al. 2010, Ben-Sahra et al. 2016) by controlling the expression of pentose phosphate pathway-related genes (Düvel et al. 2010) and glycolysis-related gene expression mediated by HIF1A (Li et al. 2014, Saxton & Sabatini 2017). In cancer cells, mTORC2 indirectly regulates c-MYC availability by promoting its mRNA translation (Masui et al. 2013) and c-MYC is a well-known key regulator, not only of metabolic reprogramming of cancer cells but also of PSC metabolism. Likewise, AKT activation in a mTORC2-dependent fashion can also modulate glucose metabolism (Deprez et al. 1997, Hagiwara et al. 2012, Roberts et al. 2013, Masui et al. 2014), thus linking energy metabolism to biosynthetic processes relevant for differentiation. Considering the metabolic resemblance between PSCs and cancer cells, it would be interesting to determine the relevance of mTOR in the regulation of the metabolic pathways relevant in PSC identity. However, although there is some literature on mTOR and pluripotency, the findings are inconsistent and mechanisms involved in regulating the expression of the pluripotency or differentiation-related networks are poorly understood.

mTOR and placenta establishment and function

The importance of nutrients such as amino acids, as well as oxygen, on embryo metabolism and redox state is clear. By promoting survival and morphological changes, adaptation to stress, regulating epigenetic and transcriptional events, signaling and biosynthetic pathways, these nutrients are able to modulate development potential (Bazer et al. 2015, Eckert et al. 2015, Gardner & Harvey 2015, Puscheck et al. 2015). Notably maternal metabolic status has a relevant role in supporting initial embryogenesis and implantation and may even affect fetal development and postnatal life (Bazer et al. 2015, Eckert et al. 2015, Puscheck et al. 2015).

In the majority of mammalian species, including rodents, ovine and porcine species, the attachment of blastocysts to the uterine luminal epithelium triggers mural TE cell differentiation into primary TE giant cells (TGCs), which in turn are able to invade the maternal tissue to reach the endometrial stroma, initiating placentation. mTOR has been shown to regulate trophectoderm cell outgrowth in the conceptuses (the embryonic/fetal and extraembryonic tissues) of these species in an amino acid- and glucose-dependent manner (Gwatkin 1966, Murakami et al. 2004, Bazer et al. 2012, González et al. 2012, Kim et al. 2013, Zeng et al. 2013, Wang et al. 2015), as well as cell migration (Correia-Branco et al. 2018) and metabolic gene expression (Rosario et al. 2019). In line with these observations, TE proliferation and migration are blocked by rapamycin in vitro (Murakami et al. 2004, González et al. 2012, Correia-Branco et al. 2018), and endometrial invasion is consequently decreased (Knuth et al. 2015). Given that amino acids are sensed by mTORC1 (Hara et al. 1998) and that rapamycin targets this complex, these studies suggest not only the prevailing role of mTORC1 rather than mTORC2 in TE cells but also that this pathway may act as an early checkpoint for development progression and successful implantation. In fact, Rictor ablation in mouse embryos results in abnormal placental histology, with smaller and thinner placentas that have an increased number of trophoblast giant cells with anomalous distribution and fewer maternal–fetal interfaces (Shiota et al. 2006).

Throughout the implantation process, endometrial nutrient supply is crucial for mTOR activity, together with TE cell crosstalk. Importantly, the availability of glucose, amino acids, fatty acids and other nutrients such as folate increases mTOR activity (Roos et al. 2009, Jansson et al. 2012, Lager et al. 2014, Rosario et al. 2017a,b), coupling the expression of placental nutrient transporters as well as their trafficking into the plasma membrane, which is also mTOR dependent (Roos et al. 2009, Rosario et al. 2011, 2013, 2016, Chen et al. 2015b). The physical link between embryonic and maternal tissues is essential to support further development events, given that the placenta is a dynamic organ and an exchange mediator between the mother and the fetus (Burton & Jauniaux 2018). Low-protein diets and caloric restriction in pregnant females result in depressed placental levels of mTOR activation, affecting nutrient transport (Rosario et al. 2011, Kavitha et al. 2014). Furthermore, placental dysfunctions, fetal growth restriction and preeclampsia can result from aberrant TE invasion and are characterized by placental insufficiency and decreased mTOR activity (Roos et al. 2007, Yung et al. 2008, Arroyo et al. 2009, Chen et al. 2015b, Rosario et al. 2017b), increased autophagy (Zhang et al. 2017b) and altered nutrient availability, including amino acid transport (Vaughan et al. 2017). On the other hand, the placentas of both obese and diabetes mellitus-affected mothers have increased mTOR activation (Gaccioli et al. 2013, Jansson et al. 2013, Capobianco et al. 2016, Villalobos-Labra et al. 2017), which is related to the increased expression and abnormal function of nutrient transporters (Gaccioli et al. 2013, Lager et al. 2014). These effects culminate in fetal overgrowth, which is typical of animal models for obesity. These conditions jeopardize fetal development and are often associated with higher mobility and mortality (Wadhwa et al. 2009, Johnsson et al. 2015). However, there are unique biochemical and morphology characteristics across placental mammals (Roberts et al. 2016), and defining a good animal model for human placental-associated pathologies is difficult. Moreover, most of the human studies are performed on term or spontaneous preterm labor placentas, but considering that it is a highly dynamic organ throughout the gestation period, some of the effects of maternal metabolic dysfunctions on mTOR status and the coupled outcomes are hard to study in vivo or to accurately capture using in vitro models.

Conclusion and future perspectives

Published literature strongly supports an important role for mTOR function, involving both mTORC1 and mTORC2, in reproductive function, preserving the necessary homeostasis for adjusted gamete production and regulating the stem cell and follicle pool, essential for sperm and oocyte production. It is also important in embryo and placenta development, as well as in PSC maintenance and differentiation (Fig. 3). However, much work is needed to uncover the specific mTOR contributions, but, as discussed here, the key aspects are related to the control of metabolic quiescence and biosynthesis. Given the role of mTOR in energy sensing and nutrient availability, future work on this key pathway could include several parallel perspectives. On the one hand, if disruption of mTOR signaling could result in subfertility, via both quiescence of the stem cell pool and its premature exhaustion, mTOR manipulation might be useful in ovarian and pre-pubertal testicular tissue preservation in oncofertility procedures, avoiding stem cell loss. This could also be extended to both embryo culture and manipulation if diapause can be pharmacologically induced in human embryos, and to control placental development to both ensure implantation and avoid overgrowth in specific cases. Finally, mTOR could also be relevant in manipulating PSC fate, with possible implications for modifications and differentiation toward specific fates.

Figure 3
Figure 3

mTOR in the reproductive cycle. The impact of the mTOR pathway on gamete production, embryo and placenta development and pluripotent stem cell maintenance and differentiation.

Citation: Reproduction 159, 4; 10.1530/REP-19-0057

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

Funding

This work was supported by Fundação para a Ciência e Tecnologia (FCT) Portugal, for PhD scholarship attributed to MIS (SFRH/BD/86260/2012). Funding was also provided by the STEM@REST Project (CENTRO-01-0145-FEDER-028871). We further acknowledge additional funding by the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme: project CENTRO-01-0145-FEDER-000012-HealthyAging2020, the COMPETE 2020 – Operational Programme for Competitiveness and Internationalisation and the Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, I.P.: project POCI-01-0145-FEDER-007440.

Author contribution statement

BC and JRS developed the original idea. BC and MIS performed literature search and analysis and provided figures. BC wrote the main version of the manuscript, revised by JRS. All authors approved the final manuscript.

Acknowledgements

The authors would like to thank the members of the Biology of Reproduction and Stem Cells research group, at the Center from Neuroscience and Cell Biology, especially Ana Sofia Rodrigues, for many discussions and constructive feedback related to this work. We would also like to thank J Saints for informal language-related suggestions.

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