Metabolic regulation is important for spermatogenesis

330 | JUNE 2012 | VOLUME 9 www.nature.com/nrurol

Health Sciences Research Centre, Faculty of Health Sciences, University of Beira Interior (CICS–UBI), 6201-506 Covilhã, Portugal (L. Rato, M. G. Alves, S. Socorro, J. E. Cavaco, P. F. Oliveira). Centre for Neurosciences and Cell Biology (CNC), University of Coimbra, 3004-517 Coimbra, Portugal (A. I. Duarte).

Correspondence to: P. F. Oliveira [email protected]

Metabolic regulation is important for spermatogenesisLuís Rato, Marco G. Alves, Sílvia Socorro, Ana I. Duarte, José E. Cavaco and Pedro F. Oliveira

Abstract | Male factor infertility is increasing in developed countries, and several factors linked to lifestyle have been shown to negatively affect spermatogenesis. Sertoli cells are pivotal to spermatogenesis, providing nutritional support to germ cells throughout their development. Sertoli cells display atypical features in their cellular metabolism; they can metabolize various substrates, preferentially glucose, the majority of which is converted to lactate and not oxidized via the tricarboxylic acid cycle. Why Sertoli cells preferentially export lactate for germ cells is not entirely understood. However, lactate is utilized as the main energy substrate by developing germ cells and has an antiapoptotic effect on these cells. Several biochemical mechanisms contribute to the modulation of lactate secretion by Sertoli cells. These include the transport of glucose through the plasma membrane, mediated by glucose transporters; the interconversion of pyruvate to lactate by lactate dehydrogenase; and the release of lactate mediated by monocarboxylate transporters. Several factors that modulate Sertoli cell metabolism have been identified, including sex steroid hormones, which are crucial for maintenance of energy homeostasis, influencing the metabolic balance of the whole body. In fact, energy status is essential for normal reproductive function, since the reproductive axis has the capacity to respond to metabolic cues.

Rato, L. et al. Nat. Rev. Urol. 9, 330–338 (2012); published online 1 May 2012; doi:10.1038/nrurol.2012.77

IntroductionA decline in male reproductive health has been observed over the last few decades in European men. Studies from Western Europe have identified a high prevalence of low sperm counts in young men (<40 years), demonstrating the impact of environmental and lifestyle factors on spermatogenesis.1–4 Several factors have been suggested to contribute to this problem, including current lifestyle in industrialized countries (diet, smoking, alcohol, drugs) and exposure to environmental chemicals, which can negatively affect testicular function to a greater degree than genetic factors.5–10 Furthermore, systemic diseases can affect the reproductive axis at multiple levels, directly or indirectly, and impair spermatogenesis irreversibly.11–13

The testicles are paired organs that essentially perform two functions: sex steroid hormone biosynthesis and production of spermatozoa. They consist of the seminiferous tubules and the intervening interstitial space. The seminiferous epithelium is compartmentalized by junctions between adjacent Sertoli cells, creating the blood–testis barrier (BTB) (Figure 1). The fully functional BTB consists of three components: an anatomical barrier restricting entry of molecules and germ cells from the basal compartment, which is in close contact with blood vessels and lymphatic endothelium, into the adluminal compartment of the seminiferous tubules; an immunological barrier that regulates the movement of

immune cells and the level of cytokines in the seminiferous epithelium; and a physiological barrier (composed of transporters and channels of the membranes) that is highly dynamic to meet the needs of both germ cells and Sertoli cells. Together, these components are essential to the function of the BTB, creating a microenvironment responsible for proper development of germ cells into fully functional sperm.14–16

Sertoli cells—often referred to as ‘nurse cells’—are responsible for providing energy and nutritional support to developing germ cells. It is imperative that germ cells receive an adequate level of energy substrates, otherwise they will degenerate and enter the apoptotic pathway.17,18 Developing germ cells have specific metabolic requirements, preferentially using lactate as a substrate for ATP production.19 Sertoli cells produce lactate via the metabolism of various substrates, preferentially glucose. Sertoli cells ensure the nutritional support of germ cells by secreting nutrients or metabolic intermediates, such as amino acids, carbohydrates, lipids, vitamins, and metal ions.20–22

The regulation of these metabolic processes is crucial and could have a direct influence on male fertility. Modulation of metabolic pathways in testicular somatic cells, especially Sertoli cells, is likely to be determined by multiple elements including metabolic substrate availability and the action of hormones and other endogenous or exogenous factors that will have a synergistic contribution to the progression of spermatogenesis. In this Review, we discuss the available literature on Sertoli

Competing interestsThe authors declare no competing interests.

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NATURE REVIEWS | UROLOGY VOLUME 9 | JUNE 2012 | 331

cell metabolism, its contribution to the formation of the mature spermatozoa and the factors that contribute to metabolic regulation.

The central role of Sertoli cellsThe BTB is one of the tightest blood–tissue barriers in mammalian tissues and the central structural element in testicular physiology,14–16 responsible for conferring polarity to Sertoli cells.16 When the BTB is dysfunctional, germ cell differentiation is arrested.23 Generation and maintenance of the BTB is assured by somatic Sertoli cells, the sustentacular cells of the seminiferous tubules,24 and its molecular composition has long been a matter of debate.16,25–28 The BTB is composed of specialized junctions between adjacent Sertoli cells, located near the basement membrane, which include tight junctions, basal ectoplasmic specializations, basal tubulobulbar complex gap junctions and desmosomelike junctions.26,27 Despite the complex composition of the BTB, it undergoes highly dynamic restructuring at specific stages of the spermatogenic cycle to allow developing germ cells to cross the BTB into the adluminal compartment.29 Throughout this process, germ cells continue to be tightly anchored to Sertoli cells via the anchoring junctions mentioned above.30 Opening and closing of the BTB is such a wellcoordinated process that even immune privilege is maintained.16

Endothelial and peritubular myoid cells (which are found outside of the seminiferous epithelium) also contribute to the selective ability of the BTB, by regulating the access of substances, including xenobiotic and vitamin Arelated compounds, into seminiferous tubules.31,32 These cells appear to be involved, together with Sertoli cells, in the metabolism and transport of retinoids into the tubules towards the germ cells.33 The compartmentalization of retinoic acid metabolism within this epithelium seems to be essential to spermatogonia proliferation and spermatogenesis.34,35

Nonetheless, seminiferous tubules are the functional units of the testes and Sertoli cells are the main structural element of the seminiferous epithelium, residing on the basement membrane.36 Both ends of each seminiferous tubule open into tubuli recti and then connect in the rete testis.37 The seminiferous tubules are lined by a complex stratified epithelium composed of Sertoli cells and germ cells (Figure 1).38 Sertoli cells occupy a volume of approximately 17–20% of the seminiferous epithelium of adult men.39 Notably, each Sertoli cell supports up to 30–50 germ cells at different stages of development,27 via numerous cytoplasmic prolongations.40 Sertoli cells form intimate associations with germ cells at different stages of their development.41 These stages represent all the steps that germinal cells undergo throughout spermato genesis.29,41,42 Mammalian spermatogenesis is a continuum of cellular differentiation with three main stages: mitotic spermato gonial proliferation and differentiation; meiotic phase; and spermiogenesis.42 This is a wellcoordinated process dependent on Sertoli cells and regulated by the hypothalamus–pituitary–testis axis.

Key points

■ Sertoli cells have multiple roles in germ cell development, ranging from physical support and immunoprotection to the supply of nutrients and other factors

■ Germ cells have specific metabolic needs, which change during their development into spermatozoa, rendering them dependent on the nurturing provided by Sertoli cells

■ Sertoli cells utilize a number of different substrates (including glucose and fatty acids) and pathways to fulfill their metabolic requirements, as well as those of developing germ cells

■ A number of hormones and factors, such as follicle-stimulating hormone, insulin, insulin growth factor-I, epidermal growth factor, paracrine factor P-Mod-S, tri-iodothyronine, basic fibroblast growth factor, cytokines, carnitine, AMP-activated protein kinase, arachidonic acid and sex steroid hormones, are known to be metabolic modulators of Sertoli cells

■ Metabolic status is central to the regulation of the energy demands of the reproductive system, and extreme metabolic disorder conditions (such as obesity) are deleterious to reproductive function

■ The reproductive axis (hypothalamus–pituitary–testis axis) is exceptionally sensitive to energetic imbalance and disturbances of this axis severely affect Sertoli cells functions

Spermatogonial stem cells, which lie at the basement membrane, replicate mitotically to both guarantee the germ cell line (spermatogonia A), and give rise to new populations (spermatogonia B) committed to differentiate and move along the seminiferous epithelium.26,42 Spermatogonia B differentiate into primary spermatocytes and then, after crossing the BTB, undergo the first division of meiosis yielding secondary spermatocytes. Round spermatids are produced through the second meiotic division. Once spermatids are formed, cell division stops and spermiogenesis starts giving rise to elongated spermatids (Figure 1). This process culminates in the release of elongated spermatids into the lumen of the tubule as immature spermatozoa (in a process called spermiation).42

The metabolic needs of germ cellsGerm cells are subjected to a number of different conditions during their development within the seminiferous tubules. Sertoli cells perform a range of functions from physical support and immunoprotection, to the supplying of nutrients and other factors in order to achieve success ful spermatogenesis.36,43,44 Furthermore, during this multifaceted process, Sertoli cells are targeted by external and internal adverse conditions, such as environ mental factors,7 hormonal deregulation,7

diseases,11 and oxidative stress,45 which might impair maintenance of the appropriate environment for proper development of germ cells.

Germ cells have peculiar nutritional requirements during spermatogenesis, switching their metabolic profile throughout development.46 Why this happens is unclear, although it is noteworthy that testes are tightly compartmentalized organs, which might restrict the availability of essential substances for germ cell energy metabolism.47 Furthermore, the testis has been reported to be a naturally oxygendeprived organ.48 Together, this may explain why germ cells utilize different metabolic pathways for energy production in their various developmental stages.46 Glycolysis has been highly conserved

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among species throughout evolution; however, many glyco lytic enzymes have testisspecific isoforms, expressed specifically or predominantly in spermatogenic cells (often during the postmeiotic phase).49

Germ cells are strictly dependent on carbohydrate metabolism, including both aerobic and anaerobic pathways.46 Spermatogonia, which lie in the basal compartment of the BTB, are supplied with nutrients from blood components and use glucose as fuel for ATP production.17 Spermatocytes are intermediate developing germ cells that may also depend on glycolysis, although the utilization of lactate by cells at these stages of development has also been reported, especially those that lie closer to the adluminal compartment.46

Although they express all enzymes of the glycolytic pathway, mature germ cells are dependent on lactate, present in extracellular medium and supplied by Sertoli cells (Figure 2).17 It has been reported that intratesticular infusion of lactate into adult crypt orchidic rat testis improves spermatogenesis.50 Lactate is also responsible for RNA and protein synthesis stimulation in spermatids18 and exerts an antiapoptotic effect on germ cells.51 Glycolytic potential in spermatids is lower than in germ cells at earlier developmental stages. Indeed, ATP levels in spermatids decrease in

response to glucose metabolism.52 However, both glycolytic and gluconeogenic pathways may be functional in round spermatids, owing to metabolic recycling of lactate to glucose6phosphate.53 Of all the germ cells, spermato zoa exhibit the highest glycolytic activity and the lowest tricarboxylic acid (TCA) cycle activity, using only glucose or fructose for their energy metab olism.46 In addition, energy production in spermatozoa is compart mentalized so that mitochondria and oxidative phosphorylation are restricted to the midpiece, while glycolysis occurs in the principal piece. The source of energy for the acrosome reaction is unclear.49

The pentose phosphate pathway is also active in germ cells, though significantly more active in spermato cytes than in spermatids, as indicated by the modulation of glucose6phosphate dehydrogenase activity in these cells.46 This pathway is required for the biosynthesis of nucleotides for RNA and the production of nicotinamide adenine dinucleotide phosphate (NADPH) and ribose 5phosphate.46 Regarding lipid metab olism, several authors have proposed that there is effective transport of long polyenes from Sertoli cells to germ cells, although it remains to be fully proved.54–56 Nevertheless, during their journey through the epididymis, rat spermatozoa undergo considerable lipid

Basement membrane

Interstitial space

Smoothendoplasmic

reticulum

Roughendoplasmic

reticulum

Myoid cell

Leydig cells

Mitochondria

Spermatogonium ASpermatogonium B

Primaryspermatocyte Secondary

spermatocyte

Spermatids

Sertolinucleus

Residualbodies

BTB

Spermatozoa

Lysosomes Sertoli cell

Bloodvessel

BTB

Figure 1 | Schematic illustration of spermatogenesis. The seminiferous epithelium is composed of Sertoli cells and developing germ cells at different stages. Leydig cells and blood vessels are located in the interstitium. Spermatogenesis is the cellular division and transformation that produces male haploid germ cells from diploid spermatogonial stem cells. Continuous sperm production is dependent upon several intrinsic (Sertoli cells and germ cells) and extrinsic (hormonal) factors. The supporting Sertoli cells adhere to the basement membrane where spermatogonia are also adherent. Spermatogonia type A divide and develop into spermatogonia type B, which enter meiotic prophase and differentiate into primary spermatocytes that undergo meiosis I to separate the homologous pairs of chromosomes and form the haploid secondary spermatocytes. Meiosis II yields four equalized spermatids that migrate toward the lumen where fully formed spermatozoa are finally released. Abbreviation: BTB, blood–testis barrier.

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remodelling and pachytene spermatocytes are known to actively metabolize fatty acids.57

The energy production capacity of the germinal cell line, which depends on the regular provision of available fuel by the somatic Sertoli cells, remains a matter of debate. It is certain, however, that the versatility of germ cells in utilizing distinct energy sources at different stages is useless in the presence of nonfunctional Sertoli cells, leading to unsuccessful spermatogenesis.

Sertoli cell metabolismThe mechanisms that regulate Sertoli cell metabolism are central to the maintenance of spermatogenesis and male fertility. Carbohydrate metabolism in Sertoli cells has some unique characteristics. For example, only 25% of the pyruvate produced from glucose in these cells is oxidized via the TCA cycle.19 Furthermore, Robinson and Fritz20 showed that cultured Sertoli cells convert the majority of glucose into lactate, which is then secreted. It has also been reported in vitro that, the pentose phosphate pathway (determined by the rate of NADPH oxidation) does not operate at its maximum rate in Sertoli cells.19,20 Finally, exogenous pyruvate is oxidized at very low concentrations by these cells during incubation with glucose.19

Sertoli cells are the major source of lactate in the testes, and several mechanisms are known to modulate production of this metabolite (Figure 2). Sertoli cells produce lactate primarily from glucose, and the ratelimiting step is the membrane passage of glucose from the extracellular space, via specific glucose transporters (GLUTs).58 Four GLUTs (GLUT1, GLUT2, GLUT3 and GLUT8) have been identified in Sertoli cells to date.59–62 However, GLUT8 is not expected to be involved in glucose transport from the extracellular milieu, since it has not been identified in the plasma membrane of Sertoli cells, but rather in the endoplasmic reticulum membrane, and thus its role in glucose uptake from extracellular space can be excluded.63,64 Lactate dehydrogenase (LDH) also has a crucial role in providing lactate to developing germ cells. The export of lactate from Sertoli cells by specific monocarboxylate transporters (MCTs) is responsible for improved lactate supply to germ cells (Figure 2).65,66

Sertoli cells have a high glycolytic activity that can be adapted to conditions of glucose deprivation, ensuring an adequate lactate concentration in the microenvironment where germ cells develop even in extreme conditions (when glucose levels are low or in the complete absence of glucose).67 In such conditions, Sertoli cells adjust their metabolism by activating specific signal transduction pathways and molecules, such as AMPactivated protein kinase (AMPK), a key mediator in cellular energy homeostasis.68 AMPK is a serine–threonine kinase that restores cellular ATP levels by switching on catabolic pathways and switching off anabolic pathways.69 Activation of AMPK increases lactate production via increased glucose uptake, and increased GLUT1 and MCT4 expression.68 This energy sensor seems to be the main activator when Sertoli cells are under stressful

conditions.70,71 Nevertheless, other metabolic sensors cannot be excluded.72

Despite being an energy substrate, glucose is not the main metabolite used for ATP synthesis in Sertoli cells, which require high energy levels to function correctly.67 Sertoli cells can maintain their viability in culture in the complete absence of glucose, still producing ATP and lactate via metabolism of lipids,73 amino acids and even glycogen.67,74,75 Xiong and collaborators73 showed that Sertoli cells preferentially use lipids as an energy source. Thus, lipid βoxidation seems to be the main metabolic pathway used by Sertoli cells to produce energy. Although Sertoli cells maintain ATP production when glycolysis is blocked, ATP synthesis decreases significantly if βoxidation is blocked.73 Interestingly (and paradoxically to their protecting and nourishing role), Sertoli cells can induce apoptosis of germ cells,76 phagocytose apoptotic spermatogenic cells, and are also responsible for endocytosis and degradation of residual bodies, converting them into lipids that are further metabolized to produce ATP.73 After engulfment of apoptotic germ cells, the expression of longchain acylCoA dehydrogenase (responsible for the catabolism of longchain fatty acids) increases significantly in mitochondria of Sertoli cells (Figure 2).73 Given that under normal physiological conditions >75% of spermatogenic cells undergo apoptosis,73 and the lipid content of residual bodies is recycled by

Sertoli cell

Germ cell

Alanine

Glucose

GlycolysisALT

Pyruvate

Pyruvate

Lactate

LDH

Lactate

Glucose

GLUT1GLUT3

MCT4

β-oxidation

Acetyl-CoATCAcycle

MCT2

Figure 2 | Schematic illustration of Sertoli cell metabolism. Sertoli cells are capable of consuming a variety of fuels including glucose, lactate and fatty acids. Sertoli cells preferentially metabolize glucose, the majority of which is converted to lactate. Lactate and pyruvate are transported out of Sertoli cells via the family of proton-linked plasma membrane transporters known as MCTs, while glucose is imported via the GLUT family of membrane proteins. Glucose enters the glycolytic pathway, which results in the production of pyruvate, which can be converted into lactate, or alanine, or be transported to the mitochondrial matrix, where it is oxidized and decarboxylated by pyruvate dehydrogenase, forming acetyl-CoA, which can enter the TCA cycle. The oxidation of these substrates is coupled with ADP phosphorylation, via the electron transport chain to form ATP. Abbreviations: ALT, alanine aminotransferase; GLUT, glucose transporter; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; TCA, tricarboxylic acid.

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Sertoli cells, there is an enormous quantity of energy reserves available to meet the energetic needs of the multi faceted Sertoli cells.

Sertoli cells can also use amino acids for energy production; oxidation of glutamine and leucine provides most of the energy required by these cells.77 Other amino acids, such as alanine and valine, also have an important role in Sertoli cell metabolism.77 Kaiser and collaborators77 suggested that low amounts of acetylCoA arising from glucose could modulate the oxidation of alanine and valine to CO2, by competing with the acetylCoA derived from these amino acids. Glucose metabolism can also stimulate the conversion of valine into lipids.77 Glutamine inhibits the oxidation of leucine, valine, and alanine, but does not alter the conversion of these amino acids into lipids. Glutamine also inhibits the incorporation of alanine into proteins.77 Alanine is the main glucogenic amino acid, since it can be converted to pyruvate that can be used as a substrate by Sertoli cells for several biochemical pathways, including the TCA cycle and gluconeogenesis. The relationship between the quantities of alanine produced and pyruvate consumed reflects the NADH:NAD+ ratio and the cytosolic redox cell status.65,66

Regulators of Sertoli cell metabolismTo date, several factors that regulate Sertoli cell metabolism and lactate production have been identified (Table 1): folliclestimulating hormone (FSH),78 insulin,78,79 insulin growth factorI,78 epidermal growth factor,80 paracrine factor PModS,81 triiodothyronine,82 basic fibroblast growth factor,83 cytokines,84 arachidonic acid,85 carnitine,86 AMPK,68 and sex steroid hormones.65,66

Gumma and collaborators87 demonstrated that FSH and insulin can affect lipid metabolism of Sertoli cells by stimulating lipid esterification. Insulin, FSH and insulin growth factorI also stimulate lactate production

by Sertoli cells, probably at the enzymatic level or via glucose transport regulation.78 Recently, it has been shown that during insulin deprivation the expression of GLUT3, LDHA, and MCT4 in human Sertoli cells decreased significantly, while GLUT1 expression increased significantly.79 FSH and interleukin 1β exert positive effects on glucose uptake, regulating glucose transporter activation or translocation in rat Sertoli cells.84 Basic fibroblast growth factor has been shown to augment lactate production through glucose uptake, via increasing GLUT1 expression and LDHA activity.88 Lactate production is also induced by epidermal growth factor in cultured Sertoli cells.80 Gallardo and collaborators68 showed that AMPK favours lactate production via an increase in GLUT1 expression and the lactate exporter MCT4. Similarly, carnitine has been described to be a positive metabolic modulator of Sertoli cell metabolism. In vitro supplementation with carnitine increased the production of both lactate and pyruvate, activity of LDH and hexose transport.86 It has also been suggested that arachidonic acid regulates lactate production by Sertoli cells, stimulating glucose uptake, LDH activity and increasing LDHA mRNA levels.85 The paracrine factor PModS appears to stimulate lactate production by Sertoli cells at various stages of pubertal development.81

Sex steroid hormones (androgens and oestrogens) also regulate Sertoli cell metabolism. Androgens are essential for the maintenance of pivotal mechanisms required for fertility,89 although few studies have reported relevant findings regarding the regulation of lactate production and metaboliterelated genes by these hormones.65,66,90–92 Recent findings, however, have shown that androgen stimulation of Sertoli cells can alter transcription of a number of metabolismrelated genes.65,66,93 We have described that 5αdihydrotestosterone (DHT) and 17βoestradiol (E2) regulate glucose uptake and lactate production in Sertoli cells from humans65 and rats.66 Moreover, DHT increased glucose uptake despite decreasing lactate synthesis, suggesting that this androgen may reduce lactate synthesis (via LDH), or transport of lactate to the extracellular medium (via MCTs).65,66 DHT drives Sertoli cells to achieve an efficient metabolic status, redirecting glucose metabolism to the TCA cycle.65,66 Results obtained in monkey epididymis (Macaca mullata) are in accordance with this hypothesis.94 Moreover, DHT was reported to stimulate succinate dehydrogenase and malate dehydrogenase activity in the epididymis of castrated animals.94 Testosterone is also involved in polyunsaturated fatty acid biosynthesis by Sertoli cells, modulating the activity of Δ5 and Δ6 desaturases.92 Experiments involving the selective ablation of the androgen receptor in mouse Sertoli cells revealed that androgens are essential to regulate glycerol3phosphate dehydrogenase expression.95 As has been described for rat Sertoli cells in vitro,66 E2 has been shown to increase GLUT3 mRNA expression in human Sertoli cells, favouring glucose uptake.65 Sertoli cells stimulated with E2 also demonstrated an increase in alanine production, indicating a lower

Table 1 | Factors regulating Sertoli cell metabolism and lactate production

Factor Expression Lactate production

GLUT1 GLUT3 MCT4 LDH

Follicle-stimulating hormone +60 /60 ND +84 +60

Insulin ND ND ND ND +78

Insulin growth factor-I ND ND ND ND +78

Epidermal growth factor ND ND ND ND +80

Paracrine factor P-Mod-S ND ND ND ND +81

Tri-iodothyronine ND ND ND ND –82

Basic fibroblast growth factor +60,88 /60,88 ND +88 +60,88

Cytokines +60 /60,84 ND +84 +60

Arachidonic acid ND ND ND +85 +85

Carnitine +120 ND ND +86 +120

AMP-activated protein kinase +68 –68 +68 ND +68

5α-dihydrotestosterone ND –65 /65,66 –65,66 –65,66

17β-oestradiol ND +65 +65 –66 /65,66

+, increase; –, decrease; /, no effect. Abbreviations: GLUT, glucose transporter; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; ND, not determined.

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redox state (higher oxidative state).65,66 Analogously, it has been described that in the lizard (Hemidactylus flaviviridis), E2 and DHT markedly suppress lactate production by Sertoli cells in a dosedependent and timedependent manner.91

Metabolism and reproductionNormal reproductive function requires an adequate nutritional intake. Conversely, extreme conditions such as caloric deprivation with weight loss, excessive food intake and obesity are deleterious for reproductive function. Recent data point toward the possible biochemical links between metabolism and reproduction.69,96,97 Although this is an issue that has received some attention, further elucidation is required.

There is a growing awareness that sex steroid hormones participate in the homeostasis of energy balance and that reproductive activity and energy metabolism are intimately related.97,98 The gonadotropinreleasing hormone (GnRH) pulse generator is exceptionally sensitive to energetic deficits, environmental contaminants and extreme exercise.99–101 In men, brief periods of fasting cause suppression of GnRH pulses, thus decreasing luteinizing hormone (LH) levels and consequently testosterone levels by downregulating the reproductive axis and therefore affecting male reproductive function.99 The hypothalamus–pituitary–testis axis is downregulated by minor energetic disturbances in men, as demonstrated by Trumble and collaborators.99 Their study revealed that LH and testosterone levels of young men were decreased, and testosterone clearance rates increased, after an evening of fasting.99 During extreme exercise, testosterone is decreased by about 55% and testicular oxidative stress is enhanced.102 Disturbances of the reproductive axis will severely affect Sertoli cell functions, since spermatogenesis is highly dependent upon both gonadotropic and androgen action.103

Androgens and oestrogens also seem to have an important role in the control of metabolic disorders. In men, E2 at physiological levels favours insulin sensitivity.104 E2 is a product of testosterone aromatization catalysed by the aromatase enzyme complex,105 and thus, a deficiency of aromatase or oestrogen receptor α leads to insulin resistance and glucose intolerance.106,107 Indeed, testosterone aromatization to E2 acting on oestrogen receptor α is essential for energy homeostasis in men.108

Energy retention in men can lead to the development of several chronic diseases, and it has been proposed that current lifestyle trends in developed countries has led to the increased incidence of multiple clinical symptoms, together termed as metabolic syndrome.109 Increased energy retention promotes adipogenesis, aromatase activity and consequent irreversible conversion of testosterone into E2, resulting in decreased testosterone and elevated oestrogen levels and directing male physiology to a hypogonadal state.110,111 Obesity and insulin resistance have been associated with hypogonadism.112 Furthermore, reduced testosterone levels promote oestrogen receptor β expression, suppressing GLUT4 expression and resulting in impaired glucose

homeostasis and insulin resistance.110 Disruption of the molecular and cellular mechanisms of reproduction may affect Sertoli cells, since they are the main target of both sex steroid hormones and FSH.103 Recently, Robeva and collaborators113 have shown that obese men with metabolic syndrome have impaired Sertoli cell function and spermato genesis. Sperm counts and sperm quality may be also affected.114

Fertility is also affected by nutrition and the availability of energy reserves. However the cellular and mol ecular mechanisms that link energy stores and reproduction, and the signals that mediate these processes, are not entirely understood. Metabolismassociated hormones, such as insulin and thyroid hormone, play crucial roles in the relationship between metabolism and reproduction.96 Triiodothyronine induces membrane hyperpolarization in Sertoli cells stimulating amino acid accumulation in immature rat testes.115 The interaction between triiodothyronine and neuro peptides is essential for the integration of metabolism and reproduction.

Three adipokines (leptin, resistin and adipo nectin) have also been associated with the link between energy reserves and reproductive function. Leptin, an adipocyte derived hormone, is pivotal in the regulation of both neuroendocrine function and fertility, stimulating GnRH secretion, gonadotropin secretion (FSH and LH) and rescuing impaired sexual function of leptindeficient mice.116 These leptindeficient mice are reproductively incompetent, but exogenous administration of leptin can reverse the situation.96 Resistin, originally described as a factor that impairs insulin sensi tivity and glucose tolerance,117 increases both basal testosterone levels and testosterone levels after human chorionic gonadotropin stimulation in a dose dependent manner.118 Adiponectin also influences the neuroendocrine axis, possibly through direct actions on the pituitary gland.119 These adipokines are involved in the control of reproductive functions at the hypothalamus–pituitary–testis axis level and may act through the AMPK system, which could be one of the signalling pathways controlling the interactions between energy balance and reproduction.

ConclusionsFormation of competent spermatozoa is an intricate and complex process initiated in the seminiferous epithelium. A critical feature of this process is the establishment of the BTB, causing physical and physiological compartmentalization of the seminiferous tubules into two different milieus that support the proliferation and differentiation of germ cells into mature spermatids. Germ cells have specific metabolic needs that render them dependent on Sertoli cells, the structural element of the seminiferous epithelium. Spermatogonia, which lie in basal compartment of BTB, use glucose as a fuel for ATP production. Moredeveloped germ cells, such as spermatids, are unable to use glucose, even though they express all enzymes of the glycolytic pathway,17 and instead utilize lactate for ATP production. Surprisingly, spermatozoa exhibit

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high glycolytic activity; chiefly metabolizing fructose but also small amounts of glucose in luminal fluid.46 The distinctive characteristics of carbohydrate metabolism in Sertoli cells allow adaptation for the progressively changing needs of germ cells. Moreover, these cells must be capable of modulating their metabolism to ensure an adequate lactate concentration in the microenvironment where germ cells develop, even in conditions of glucose limitation. It has been suggested that production of lactate may be derived not only from carbohydrates, but also from amino acids or glycogen metabolism, although these processes are not entirely understood.

Further knowledge of these biochemical mechanisms and of the influence of whole body metabolic status on reproduction, as well as how this regulation contributes to male reproductive function, is essential.

Review criteria

Following the hypothesis that metabolic regulation of seminiferous epithelium cells could control spermatogenesis, several web-based libraries (PubMed, ISI Web of Knowledge and Scholar Google) were comprehensively searched for papers published between 1950 and February 2012 using the terms “environmental effects spermatogenesis”, “environmental toxicants male reproductive function”, “lifestyle effects”, “blood-testis barrier function”, “seminiferous epithelium”, “spermatogenesis”, “Sertoli cells”, “germ cells metabolism”, “lactate germ cells”, “Sertoli cell metabolism”, “metabolic modulation Sertoli cells”, “energy balance reproduction” and “reproductive system and energy”. Literature database results were screened for relevance to this Review. The abstracts of relevant titles were read and all studies that potentially met the inclusion criteria were selected.

1. Jorgensen, N. et al. Regional differences in semen quality in Europe. Hum. Reprod. 16, 1012–1019 (2001).

2. Jorgensen, N. et al. East-West gradient in semen quality in the Nordic-Baltic area: a study of men from the general population in Denmark, Norway, Estonia and Finland. Hum. Reprod. 17, 2199–2208 (2002).

3. Fernandez, M. et al. Semen quality and reproductive hormone levels in men from Southern Spain. Int. J. Androl. 35, 1–10 (2012).

4. Nordkap, L., Joensen, U. N., Jensen, M. B. & Jorgensen, N. Regional differences and temporal trends in male reproductive health disorders: Semen quality may be a sensitive marker of environmental exposures. Mol. Cell. Endocrinol. http://dx.doi.org/10.1016/ j.mce.2011.05.048.

5. Bustos-Obregón, E. & Hartley, B. Ecotoxicology and testicular damage (environmental chemical pollution): a review. Int. J. Morphol. 26, 833–840 (2008).

6. Mathur, P. P. & D’Cruz, S. C. The effect of environmental contaminants on testicular function. Asian J. Androl. 13, 1–7 (2011).

7. Sharpe, R. M. Environmental/lifestyle effects on spermatogenesis. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 365, 1697–1712 (2010).

8. Goulis, D. G. & Tarlatzis, B. C. Metabolic syndrome and reproduction: I. testicular function. Gynecol. Endocrinol. 24, 33–39 (2008).

9. Mah, P. M. & Wittert, G. A. Obesity and testicular function. Mol. Cell. Endocrinol. 316, 180–186 (2010).

10. Bonde, J. P. & Storgaard, L. How work place conditions, environmental toxicants and lifestyle affect male reproductive function. Int. J. Androl. 25, 262–268 (2002).

11. Suehiro, R. M. et al. Testicular Sertoli cell function in male systemic lupus erythematosus. Rheumatology 47, 1692–1697 (2008).

12. Karagiannis, A. & Harsoulis, F. Gonadal dysfunction in systemic diseases. Eur. J. Endocrinol. 152, 501–513 (2005).

13. Sartorius, G. A. & Handelsman, D. J. in Andrology: Male Reproductive Health and Dysfunction (eds Nieschlag, E., Behre, H. M. & Nieschlag, S.) 339–364 (Springer, Berlin, 2010).

14. Su, L., Mruk, D. D. & Cheng, C. Y. Drug transporters, the blood-testis barrier and spermatogenesis. J. Endocrinol. 208, 207–223 (2011).

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AcknowledgementsThis work was supported by the Portuguese “Fundação para a Ciência e a Tecnologia”—FCT (PTDC/QUI-BIQ/121446/2010) co-funded by FEDER via Programa Operacional Factores de Competitividade—COMPETE/QREN. L. Rato (SFRH/BD/72733/2010), M. G. Alves (SFRH/BPD/80451/2011) and A. I. Duarte (SFRH/BPD/26872/2006) were financed by FCT. P. F. Oliveira was financed by FCT through FSE and POPH funds (Programa Ciência 2008).

Author contributionsL. Rato and M. G. Alves researched data for the article. L. Rato, M. G. Alves and P. F. Oliviera contributed substantially to discussion of content, writing and reviewing/editing the manuscript before submission. S. Socorro, A. I. Duarte and J. E. Cavaco contributed substantially to discussion of content and reviewing/editing the manuscript before submission.

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