Peroxisome proliferator-activated receptor gamma signaling in human sperm physiology.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the PPARs, which are transcription factors of the steroid receptor superfamily. PPARγ acts as an important molecule for regulating energy homeostasis, modulates the hypothalamic-pituitary-gonadal (HPG) axis, and is reciprocally regulated by HPG. In the human, PPARγ protein is highly expressed in ejaculated spermatozoa, implying a possible role of PPARγ signaling in regulating sperm energy dissipation. PPARγ protein is also expressed in Sertoli cells and germ cells (spermatocytes). Its activation can be induced during capacitation and the acrosome reaction. This mini-review will focus on how PPARγ signaling may affect fertility and sperm quality and the potential reversibility of these adverse effects.

INTRODUCTION

Peroxisome proliferator-activated receptor gamma (PPARγ) was originally named for its ability to induce hepatic peroxisome proliferation in mice in response to xenobiotic stimuli.1 It belongs to the nuclear hormone receptor superfamily of ligand-activated transcription factors. The PPAR family consists of three primary subtypes, PPARγ, PPARβ/δ and PPARγ, which are encoded by separate genes.2 These receptors play a central role in the physiological processes that have an impact on lipid homeostasis, inflammation, adipogenesis, reproduction, wound healing, and carcinogenesis.345 PPARγ is also implicated in a wide variety of cellular functions and regulates the expression of gene networks required for cell proliferation, differentiation, morphogenesis and metabolic homeostasis. It is possible to hypothesize that PPARγ potentially activates lipogenic genes and adipocyte differentiation.678 PPARγ is highly expressed in adipose tissue9 and it is necessary for adipocyte differentiation and transformation of many nonadipogenic cell lines into adipocyte-like cells. PPARγ is also an important transcriptional regulator that modulates cellular glucose and lipid metabolism.10 Intensive studies and compelling evidence have demonstrated that PPARγ is a link between energy metabolism and reproduction, as in male infertility because of obesity, which is frequently associated with insulin resistance.11

Thorough studies have demonstrated a close link between energy status and reproductive functions.12 In mice, loss of the PPARγ gene in oocytes and granulosa cells results in impaired fertility.13 Moreover, Aquila et al. have demonstrated that human spermatozoa express PPARγ protein and investigated its functions.14 Recently, repetition of thorough studies have indicated that sperm cells express various receptor types,1516 and also produce their ligands, suggesting that an autocrine short loop may modulate sperm cell's function independently by systemic regulation.1718 Nevertheless, it is necessary for spermatozoa to regulate their metabolism to affect the changes in signaling pathways encountered during their life. However, the mechanisms underlying the signaling events associated with the change in sperm energy metabolism are, to date, poorly understood.

Here, we will briefly review the mechanisms of sperm physiology, determining whether PPARγ signaling affects sperm capacitation and the possible targets of therapy of male infertility. PPARγ agonists may be used in artificial insemination or other biotechnologies, including cryopreservation.

EXPRESSION AND PUTATIVE ROLES OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA SIGNALING IN THE REPRODUCTIVE TISSUES

Hypothalamic-pituitary-gonadal axis

Early studies elucidated that adenoma cells can suppress the proliferation of pituitary cells,19 and the administration of thiazolidinediones (TZDs) inhibits the development of pituitary adenomas in mice and man. Furthermore, in the pituitary gland of mice, the expression of PPARγ is reduced by 54% after 24 h of food restriction.20 In the hypothalamus, PPARγ regulates a variety of molecules involved in energy homeostasis,20 mainly playing a role in temperature regulation through its natural ligand 15-deoxy-delta12, 14-prostaglandin J2 (PGJ2), which is secreted into cerebrospinal fluid.21 It is still unclear whether the effect of PPARγ on reproductive function is mediated by this signal pathway. Some pituitary tumors secrete hormones such as prolactin (PRL) and growth hormone (GH). In most of PRL- and GH-secreting pituitary tumors, these hormones control tumor growth or induce tumor shrinkage.22 Moreover, pituitary PPARγ is abundantly expressed in human PRL-, GH-secreting, and nonfunctioning pituitary tumors.19 Conditional knockout of PPARγ in pituitary gonadotrophs causes an increase in luteinizing hormone levels in female mice, a decrease in follicle-stimulating hormone (FSH) in male mice, and a fertility defect in knockout mice characterized by reduced litter size.23 Moreover, it has been reported that PPARγ functions are regulated by FSH through mitogen-activated protein kinase (MAPK) signaling pathways ().24 Thus, it is suggested that PPARγ signaling participates in the regulation of pituitary hormones.

PPARγ functions in hypothalamic-pituitary-gonadal axis. Pulsatile GnRH production signals gonadotroph cells in the anterior pituitary to produce FSH and LH that then act on the testis to regulate spermatogenic potential. FSH up-regulates the expression of PPARγ through MAPK signaling pathways while LH inhibits the function of PPARγ via various pathways. High expression of testosterone suppresses the secretion of LH by negative feedback, providing a relatively persistent high-expression of PPARγ. PPARγ: peroxisome proliferator-activated receptor gamma; FSH: follicle-stimulating hormone; LH: luteinizing hormone; MAPK: mitogen-activated protein kinase.

In the testis, the PPARs are expressed in both somatic and germ cells.25 PPARα and PPARβ are widely expressed in the interstitial Leydig cells and the seminiferous tubule cells (Sertoli and germ cells),26 whereas, PPARγ is believed to be restricted to Sertoli cells.27 Sertoli cells are the first cells to differentiate recognizably in the undifferentiated fetal gonad, an event, which enables seminiferous cord formation, prevention of germ-cell entry into meiosis, differentiation, and function of Leydig cells.28 During puberty, Sertoli cells also play vital roles in supporting spermatogenesis. Without the physical and metabolic support of Sertoli cells, germ-cell differentiation, meiosis and transformation into spermatozoa would not occur.29 Moreover, Thomas et al. have recently detected PPARγ mRNA in the germ cells (spermatocytes).30 It may be that PPARγ signaling regulates the pattern of expression of key lipid and glucose metabolic genes in the Sertoli cells.

Spermatogenesis

Spermatogenesis is the successful transformation of round spermatids into the complex structure of the spermatozoon ().31 However, the physiological demands of reproduction are energetically costly and mating behavior and physiological responses are inhibited when fuel reserves or food intake is limited. Indeed, inadequate metabolic fuel utilization is the common factor of nutritional infertility.32 Of the sources of stored energy that can be tapped for fuel reproductive energy requirements, the largest depot is white adipose tissue (WAT), which is primarily composed of white adipocytes that store lipid fuels as triacylglycerols.333435 Epididymal WAT (EWAT) is necessary for normal spermatogenesis and could produce a locally acting factor responsible for maintaining spermatogenesis since a decrease in EWAT causes a disturbance in spermatogenesis.3436 However, removal of comparable amounts of WAT from other sites (inguinal) shows no effect, disproving the idea that the effect is due to a decreased energy supply or the need for some minimal amount of fat.37 It has been suggested that it might be due to the presence of a local, but currently unidentified, growth or nutritive factor from EWAT that promotes spermatogenesis. PPARγ, known as one of the master regulators in adipogenesis, is also developmentally expressed both in differentiating germ and Sertoli cells,2730 where it is involved in regulating the patterns of expression of key lipid metabolic genes in Sertoli cells.30 It is also indicated that PPARγ signaling plays an important role in spermatogenesis.30

Expression patterns of PPARg shows in the testis. (a) In the testis, PPARγ protein is detected at high expression in Sertoli cells and weak expression in spermatocytes. The names of cells expressing PPARγ are underlined. (b) PPARγ forms obligate heterodimers with RXRγ for regulation of lipid metabolic target genes, providing energy for spermatogenesis. PPARγ: peroxisome proliferator-activated receptor gamma; RXRγ: retinoid X receptor gamma.

Mechanisms of peroxisome proliferator-activated receptor gamma signaling in spermatogenesis

The PPARs form obligate heterodimers with the retinoid X receptors (RXRs) to produce functional transcription factors that are involved in transactivation of several key genes during energy homeostasis and cellular differentiation ().30383940 TZDs, the synthetic ligands of PPARs, have been demonstrated to modify PPAR-mediated transcriptional activation of a number of key genes involved in energy homeostasis.41 Furthermore, Thomas et al. have demonstrated that PPAR and RXR transcripts encoding members of the PPAR and RXR nuclear receptor family reach maximum levels of expression in the germ cells during the early meiotic stages of spermatogenesis.30 PPARγ levels peak at a slightly later stage of spermatogenesis in leptotene/zygotene spermatocytes, concomitant with increased levels of RXRβ and RXRγ expression. PPARγ/RXRγ heterodimeric transcription factor complexes, the predominant transcripts expressed in mature Sertoli cells, up-regulate lipid metabolic target genes in Sertoli cells, providing them with enough energy to support spermatogenesis. Infertility occurs if there is an interruption of the spermatogenic program.4243 In addition, male fertility can be compromised by inactivation of genes involved in lipid metabolism.44 In summary, except for its role in spermatogenesis, PPARγ participates in fertilization by supporting energy provision.

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA ACTION IN FERTILIZATION

Roles of peroxisome proliferator-activated receptor gamma in fertilization

Fertilization is a complex program of biochemical changes that spermatozoa undergo in the female reproductive tract. Once capacitated, the spermatozoon can bind to the zona pellucida of the oocyte and undergo the acrosome reaction (AR), a process that enables sperm penetration and fertilization of the oocyte.45 Some intracellular changes, including an increase in cholesterol efflux, a rise in membrane fluidity, an increase in intracellular Ca2+ concentration,4647 and actin polymerization,4849 have been considered to be the acceptable markers of capacitation. PPARγ agonist was able to elevate the functional maturation of sperm by evaluating its action on capacitation.50 Recent research has demonstrated that PPARγ is expressed by ejaculated spermatozoa of humans and pigs, improving their motility, capacitation, AR, survival and metabolism.1450

Roles of peroxisome proliferator-activated receptor gamma in infertility

Infertility is the inability to conceive after 12 months of regular, unprotected intercourse,51 which is a problem of public health importance in China and many other developing nations because of its high prevalence and its serious social implications for affected couples and families. Epidemiological studies have confirmed that infertility affects approximately 5% of newly married couples in Shanghai, China. Under infertility treatment, about 60% of couples subsequently have a higher chance of having children than the untreated.52 Recently, reports have asserted that sperm concentrations have been identified a potential decline over the past several decades, which may result in the decline in male fertility; however, the causes and extent of declining sperm quality and fertility remain unknown in most cases. Beyond the growing burden of disease, male infertility, associated with a high cost of care, generates significant psychosocial and marital stress. In addition, paternal health cues can be passed to the next generation, with male age associated with an increase in autistic spectrum disorders53 and environmental exposures associated with increases in incidences of childhood diseases.5455 Likewise, there is now evidence that paternal infertility may be transferred to the offspring, including metabolic diseases.56 As a result, several factors relating to general health and well-being, such as diet,57 exercise,58 obesity,59 and psychological stress,6061 have been extensively studied for their effects on male reproductive potential. Special attention has been paid to the connections between obesity and sperm function. It is of great importance to find out the causes of declining sperm quality and fertility, which adversely affect human reproduction. It is a matter of great concern triggering large-scale studies into its causes and possibilities for prevention.

There is now emerging evidence that male obesity has a negative impact on male reproductive potential not only by reducing sperm quality, but in particular by altering the physical and molecular structure of testicular germ cells and ultimately mature spermatozoa.59 Meanwhile, hyperinsulinemia and hyperglycemia are common in obese individuals and are constant confounding factors in many rodent studies of male obesity.626364 Apart from these, the fuel sensors glucose, insulin6566 and leptin676869 are known to be directly involved in the regulation of fertility at each level of the hypothalamic-pituitary-gonadal (HPG) axis.70 The discovery of the PPAR family of transcription factors has revealed a link between lipid or glucose availability and long-term metabolic adaptation.70 Historically, the roles of PPARγ have been associated with preadipocyte expansion and differentiation.71 PPARγ mainly plays key roles in the regulation of cellular lipid metabolism, redox status and organelle differentiation in adipose tissue and other organs such as the prostate.7273 Therefore, it remains plausible that PPARγ participates in the regulation of male reproductive function, by reducing sperm motility and inducing male infertility.

Roles of peroxisome proliferator-activated receptor gamma in sperm capacitation and sperm metabolism

Sperm capacitation is an intricate program in which a myriad of events take place with the result that spermatozoa can penetrate and fertilize the oocyte. The bioenergetics of sperm capacitation is poorly understood despite its fundamental role in sustaining the biochemical and molecular events occurring during gamete activation. Adenosine triphosphate is synthesized by spermatozoa through either aerobic or anaerobic metabolic pathways. Santoro et al. demonstrated that in the majority of spermatozoa, PPARγ was expressed in the apical region of the head, in the subacrosomial region and prevalently in the midpiece, while the signaling was almost absent from the tail. However, in capacitated spermatozoa, the location of the receptor mirrors that observed in uncapacitated sperm cells.50 It has been confirmed that PGJ2, an agonist of PPARγ, increases the viability of spermatozoa, whereas all these events are reduced by the irreversible PPARγ antagonist GW9662,50 confirming the involvement of PPARγ in sperm viability. Meanwhile, PPARγ antagonist GW was able to attenuate the functional maturation of spermatozoa by evaluating its action on capacitation which has been correlated with functional and biochemical changes in sperm cells, including cholesterol efflux and tyrosine phosphorylation of sperm proteins.50 Hence, it is reasonable to believe that PPARγ participates in capacitation by glucose metabolism or other metabolic pathways, and increases the motility of capacitated spermatozoa.

Glucose metabolism is a critical pathway that can produce sufficient energy for the sustenance of life. Given the beneficial effects of PPARγ ligands in therapies aimed at lowering glucose levels in type 2 diabetes, a role for PPARγ in glucose metabolism has been explored.7475 The effect of glucose on the fertilizing ability of spermatozoa appears to be mediated by the pentose phosphate pathway (PPP).76 Metabolism of G-6-P through the PPP yields much more nicotinamide adenine dinucleotide hydrogenase (NADPH) than glycolysis and TCA cycle, and NADPH acts as a hydrogen donor in many chemical reactions in vivo.77 G6PDH is a key rate-limiting enzyme in this metabolic pathway and has been shown to be functional in human spermatozoa.78 PPARγ is able to modulate in a dose-dependent way the activity of G6PDH in spermatozoa.50 Meanwhile, PPARγ has the potential to increase peripheral tissue sensitivity to insulin, thereby improving insulin resistance. Insulin resistance appears to negatively affect the sperm quantity and quality.79 Moreover, insulin is a known mediator and modulator of the HPG axis, contributing to the regulation of male reproductive potential and overall wellbeing.7980 Its disruption of the HPG axis can render patients hypogonadal. It has been shown that hyperinsulinemia is associated with increased seminal insulin concentrations, which may negatively impact male reproductive function in obesity.80

CROSS-TALK BETWEEN PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA AND SIGNALING TRANSDUCTION PATHWAYS

Peroxisome proliferator-activated receptor gamma and the PI3K signal transduction pathway

Once an insulin receptor substrate (IRS) combine with its catalytic subunits, the IRS catalyzes the phosphorylation of membrane phosphatidylinositol (PI). PI3K, which has been shown to be active in human spermatozoa,81 is important in a wide variety of cellular processes in which PI3K activation leads to production of 3’- phosphoinositide second messengers, such as PI- 3,4,5-trisphosphate, which activate a variety of downstream cell survival signals.82 Accumulation of PI 3,4,5-trisphosphate in the membrane recruits a number of signaling proteins containing pleckstrin homology domains, including AKT and PDK1.8283 On recruitment, AKT becomes phosphorylated and activated by a series of enzymes, kinases and transcription factors downstream, and yields a variety of biological functions, including intracellular trafficking, organization of the cytoskeleton, cell growth and transformation, and prevention of apoptosis.8485 Interestingly, AKT is able to stimulate the metabolism of glucose through activation of AS160, the substrate of AKT, and promotes transposition of GLUT4 and absorption of glucose into muscle cells. PPARγ activation has been reported to regulate components of the PI3K signaling cascade in various cell types,86 enhancing the sensitivity of insulin. Elevation of Glut4 and PPAR gene expression in parallel with glucose uptake has been confirmed by in vitro glucose uptake activity.87 There is evidence that increasing doses of PPARγ agonists increase Akt1/Akt2/Akt3 significantly, whereupon. AKT, the major downstream gene of PI3K signal transducer, is fully activated.50

Peroxisome proliferator-activated receptor gamma and the leptin signal transduction pathway

In addition to its role in metabolic control, leptin has pivotal roles in reproduction88 and neuroendocrine signaling.8990 Various pieces of evidence have pointed to a direct role of leptin in the control of male reproduction.91929394 In particular, ob/ob male mice (lacking functional leptin) or db/db male mice (lacking functional leptin receptor) are infertile and fail to undergo normal sexual maturation.95 In human, leptin is expressed in the seminiferous tubules9697 and in seminal plasma9899 while the leptin receptor is found in the interstitium, primarily in the Leydig cells.97 Worthy of note, Camiña et al. first proposed that human leptin is present in seminal fluid, with at least two charge variants and no binding proteins, the most likely source being either the seminal vesicles or prostate.99 Hence, it is reasonable to speculate that leptin has a direct (paracrine, autocrine or both) effect on epithelial cells of the male accessory genital glands, and on the spermatozoa via sperm leptin receptors.100 OBR, a single membrane-spanning glycoprotein, belonging to the class I cytokine receptor superfamily, shares sequence homologies for interaction with Janus kinase (JAK) as well as STATs.101 Nonetheless, PPARγ, whose promoter region is rich in multiple Stat5 DNA binding consensus sequences, is downstream of the JAK/STAT signaling pathway,102 suggesting that expression of this gene is regulated by the JAK/STAT pathway.

Experimental studies have shown that leptin treatment results in a significant increase in cholesterol efflux from and protein tyrosine phosphorylation of pig spermatozoa, stimulates pig sperm acrosin activity,103 two events associated with capacitation.104105106 Compelling evidence suggests that leptin has a direct inhibitory effect on rosiglitazone-induced adipocyte differentiation and PPARγ expression, in which ERK1/2 MAPK and JAK/STAT1 signaling pathways are involved.107108 Several studies have supported a relationship between increased leptin production and regulation of reproductive function. Indeed, leptin plays a critical role at every level of the HPG axis in males. Most obese male mice become insensitive to increased endogenous leptin production and develop functional leptin resistance.109110 This deregulation of leptin signaling might result in abnormal endocrine and reproductive functions with altered leptin dynamics, and may contribute to male infertility in different ways, leading to hypogonadism.111 Therefore, PPARγ agonists may enhance the sensitivity of insulin, acting as a potential therapy for hypogonadism.

SUMMARY

Peroxisome proliferator-activated receptor gamma may play a key role in linking lipid metabolism and reproduction in general. Energy from glucose and fat metabolism mediated by PPARγ signaling is required for sperm physiology, affecting male fertility. These recent experiments raise several questions. One question concerns PPARγ agonist activation of related metabolic pathways. Owing to the role of PPARγ in sperm capacitation, the use of its agonists may be considered a strategy in artificial insemination or other biotechnologies. Another question is whether the positive effects of PPARγ agonists are due to a direct effect on the testis or a positive effect on glucose homeostasis. Further experiments are needed to increase our knowledge of the way in which PPARγ signaling maintains sperm viability.

AUTHOR CONTRIBUTIONS

LL drafted the manuscript. HX and JCC participated in the design of the study and helped draft the manuscript. CZ, YHZ, MMC and YQ helped draft the manuscript. MJ conceived of the study, and participated in its design and coordination and helped draft the manuscript. All authors read and approved the final manuscript.