Autophagy in male reproduction

ABSTRACT

Autophagy is a fundamental process that exists in all eukaryotic organisms, with a primary function of catabolizing undesirable components to provide energy and essential materials. Increasing evidence illustrates that autophagy is invovled in a broad range of cellular events within the male reproductive system. In the process of spermatogenesis, autophagy is crucial for the formation of specific structures that guarantee successful spermatogenesis, as well as for the degradation of certain constituents. The underlying connections between autophagy and androgen binding protein, lipid metabolism and testosterone biosynthesis would increase our understanding of male testicular endocrinology. Moreover, cumulative studies reveal that autophagy is a double-edged sword when the organism suffers from endocrine disrupting chemicals. This review contains a collection of the current literature concerning the above aspects of autophagy, which may provide insights for future study and exploration.

Abbreviations: 3-MA: 3-methyladenine; ABP: androgen-binding protein; AKT: protein kinase B; AMPK: adenosine monophosphate-activated protein kinase; ART: assisted reproductive technologies; Atg: autophagy-related gene; CE: cholesteryl ester; CL: corpus luteum; CQ: chloroquine; CYP11A1: cholesterol side chain cleavage enzyme; CytC: cytochrome C; DEHP: di-2-ethylhexyl phthalate; DFCP1: double FYVE-containing protein 1; EDCs: endocrine-disrupting chemicals; ERK1/2: extracellular signal-regulated kinase 1/2; ES: ectoplasmic specialization; FC: free cholesterol; FIP2000: focal adhesion kinase family interacting protein of 200kDa; FSH: follicle stimulating hormone; HDL: high-density lipoprotein; IVF: in vitro fertilization; LC3: microtubule-associated protein light chain 3; LD: lipid droplet; LH: luteinising hormone; MC-LR: microcystin-LR; MEFs: mouse embryonic fibroblast cells; MT: microtubule; mtDNA: mitochondrial DNA; mTOR: mammalian target of rapamycin; NHERF2: Na⁺/H⁺ exchanger regulatory factor 2; NMR: naked mole-rat; PCD: programmed cell death; PDLIM1: PDZ and LIM domain 1; PGCs: primordial germ cells; PGF_2α: prostaglandin F_2α; PI3K: phosphatidylinositol-3-kinase; PI3P: phosphatidylinositol-3-phosphate; ROS: reactive oxygen species; SCG10: superior cervical ganglia protein 10; SR-BI: scavenger receptor class B, type I; StAR protein: steroidogenic acute regulatory protein; TC: total cholesterol; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase mediated dUTP nick end labeling; ULK1: mammalian uncoordinated-51-like kinase 1; WIPI: WD-repeat domain phosphoinositide-interacting.

KEYWORDS:

• Autophagy
• EDCs
• infertility
• reproduction
• spermatogenesis
• testosterone

Introduction

Today infertility affects approximately 10–15% of men of reproductive age worldwide, and male factors are account for 50% of voluntarily childless couples with abnormal semen parameters (Sharlip et al. 2002; Luke 2017). Among the infertile males, 30–40% cases are diagnosed with idiopathic male infertility with no identified etiological factors (Jungwirth et al. 2012), which may be associated with the unelucidated spermatogenic mechanisms.

Table 1. Mutant animal models and the role of autophagy in main studies of this review.

Animal models	Structure or procedure	References	Involvement of autophagy
Atg5 or Atg7 knockout in mouse Sertoli cells	Ectoplasmic specialization	Liu et al. 2016	Degradation of a negative cytoskeleton organization regulator, PDLIM1, to maintain normal cytoskeletal organization
Atg7 knockout in mouse germ cells	Acrosome biogenesis	Wang H. et al. 2014	Fusion and transportation of Golgi apparatus – derived proacrosomal granules
Atg7 knockout in mouse germ cells	Flagella biogenesis and cytoplasm removal	Shang et al. 2016	Degradation of a negative cytoskeleton organization regulator, PDLIM1, to maintain normal cytoskeletal organization
siAtg7 in rat primary Leydig cells	Testosterone biosynthesis	Ma et al. 2018	Degradation of intracellular LDs/TC to provide substrate for synthesizing testosterone
Atg5 or Atg7 knockout in mouse Leydig cells	Testosterone biosynthesis	Gao et al. 2018	Degradation of a negative SR-BI regulator, NHERF2, to facilitate the uptake of cholesterol for synthesizing testosterone
siRNA for Atg7 or MTOR in rat primary Sertoli cells	ABP metabolism	Ma et al. 2015	Degradation of ABP, which is selectively regulated by testosterone

Autophagy, also interpreted as self-eating, is an evolutionarily conserved cellular process found in all eukaryotic organisms (Levine and Klionsky 2004). Three main types of autophagy, macroautophagy, microautophagy and chaperone-mediated autophagy, are commonly recognized, with different molecular mechanisms (Klionsky 2005). Microautophagy is a process that engulfs the cytoplasm directly at the lysosomal membrane through invagination and/or septation. In comparison, the most prevalent type, macroautophagy, involves the formation of a double-membrane vesicle that sequesters the cytoplasm. Then, the completed vesicle, termed the autophagosome, fuses with the lysosome for subsequent degradation. In contrast to previous studies, chaperone-mediated autophagy targets and translocate the selective proteins to the lysosomal lumen via chaperone proteins (Klionsky 2005). The autophagy discussed here is macroautophagy unless otherwise indicated. Autophagy can serve as a scavenger by degrading the dysfunctional or unnecessary cytoplasmic components to provide energy and building materials to maintain cellular homeostasis. It can be triggered by a variety of internal or external stimuli through single or multiple signal pathways. For instance, glucose withdrawal or mitochondrial dysfunction induces adenosine monophosphate-activated protein kinase (AMPK), which negatively manipulates the mammalian target of rapamycin (mTOR) signaling cascade (Jung et al. 2010). In addition, hypoxia, reactive oxygen species (ROS) and the accumulation of unfolded proteins can also provoke autophagy as a cytoprotective mechanism in unfavorable circumstances (Lin and Baehrecke 2015). It has been reported that autophagy is also involved in various pathological situations, such as cancer, infection, neurodegenerative diseases, and cardiovascular diseases (Choi et al. 2013).

Autophagosome biogenesis begins with the initiating phagophore membrane, which originates from multiple sources, such as the endoplasmic reticulum (Weidberg et al. 2011). Then, the activated UNC-51-like kinase 1 (ULK1, a yeast ATG1 homolog) complex, which consists of ULK1, ATG13, ATG101 and focal adhesion kinase family interacting protein of 200kDa (FIP200), associates with the initiating phagophore membrane (Weidberg et al. 2011). The class Ⅲ phosphatidylinositol-3-kinase (PI3K) complex (including BECN1/ATG6, VPS34, VPS15 and ATG14) is successively recruited to the phagophore membrane, and it phosphorylates membrane-located phosphatidylinositol to form phosphatidylinositol-3-phosphate (PI3P) (Lin and Baehrecke 2015). PI3P recruits effector proteins such as double FYVE-containing protein1 (DFCP1) and WD-repeat domain phosphoinositide-interacting (WIPI) to this membrane (Weidberg et al. 2011). Subsequently, the phagophore membrane undergoes elongation, which requires two ubiquitin-like conjugation systems: the ATG5-ATG12 conjugation system and the microtubule-associated protein light chain 3 (LC3)/ATG8 conjugation systems. ATG12 is connected to ATG5 by ATG7 and ATG10 enzymes, and then the ATG12-ATG5 conjugate interacts with ATG16 to form a complex. The precursor of LC3 is processed by the protease ATG4 to generate the LC3-I form. Then, LC3-I is conjugated with phosphatidylethanolamine (PE) by the ATG7 and ATG3 enzymes to generate the LC3- II form, which plays an essential role in the formation of double-membrane autophagosomes (Mizushima et al. 2010) . The double-membrane autophagosome sequesters the targeted substrates, while its outer membrane subsequently fuses with the lysosome to form an autolysosome, in which the engulfed contents are degraded by lysosomal acid hydrolases for recycling (Ravikumar et al. 2010). Several agents act on different targets in this process (Kabeya et al. 2000; Jung et al. 2010; Ravikumar et al. 2010; Choi et al. 2013). (Figure 1)

Figure 1. The biogenesis of the autolysosome. (1) The activated UNC-51-like kinase 1 (ULK1) complex is recruited to the initiating phagophore membrane. (2) The class Ⅲ phosphatidylinositol-3-kinase (PI3K) complex is attached with the phagophore membrane and phosphorylates phosphatidylinositol to generate phosphatidylinositol-3-phosphate (PI3P). (3) Effector proteins of PI3P, like double FYVE-containing protein1 (DFCP1) and WD-repeat domain phosphoinositide-interacting (WIPI), are linked with the membrane. (4) The elongation phase of the membrane requires the ATG5-ATG12 conjugation system and the microtubule-associated protein light chain 3 (LC3)/ATG8 conjugation system, then a double-membrane autophagosome is formed. (5) The mature autophagosome is fused with the lysosome to develop an autolysosome. Several agents act on different sites of the autolysosome biogenesis. For instance, 3-methyladenine (3-MA) and wortmannin suppress the formation of autophagy by inhibiting vacuolar protein sorting 34 (VPS34) of the ULK1 complex, chloroquine (CQ) impairs autophagosome-lysosome fusion and lysosomal degradative activity, and bafilomycin A1 inhibits vacuolar type proton ATPase and the fusion between autophagosomes and lysosomes. Conversely, rapamycin serves as an agonist of autophagy through its inhibition of mammalian target of rapamycin (mTOR, which is part of the mTOR complex), since the mTOR complex is a negative regulator of the ULK1 complex.

Depending on the selective substrates, such as mitochondria, lipid droplets, ribosomes, portions of the endoplasmic reticulum being used for catabolism, mitophagy, lipophagy, ribophagy and reticulophagy have also been reported (Tolkovsky 2009; Liu and Czaja 2013; Weckman et al. 2014). Recent research has shown that lipophagy is involved in the testosterone biosynthesis and likely supplies endogenous energy for the development of germ cells (Ahmed et al. 2016; Gao et al. 2018; Ma et al. 2018; Tarique et al. 2019). In addition, mitophagy may have an antiapoptotic role in the ethanol-treated rat Sertoli cells (Eid et al. 2012; Horibe et al. 2019), and it possibly participates in the elimination of paternal mitochondria after fertilization among different species (Sato and Sato 2011; Al Rawi et al. 2012; Luo et al. 2013; Politi et al. 2014; Rojansky et al. 2016; Song et al. 2016). Herein we review relevant literature concerning the role of autophagy in the male spermatogenesis and testicular endocrinology, as well as in the existence of endocrine-disrupting chemicals.

Autophagy in the process of spermatogenesis

Spermatogenesis is a complex process in which successive cellular events occur sequentially in specific regions of the testis. It includes the mitosis of the spermatogonia, two meiotic divisions of the spermatocytes, spermiogenesis and spermiation. During this process, the germ cells are transported across the seminiferous epithelium by the Sertoli cells (Qian et al. 2014). The seminiferous epithelium exhibits tight junctions, gap junctions and the testis-unique junctions such as the ectoplasmic specialization (ES). ES, an actin microfilament-rich anchoring junction, includes the basal ES and apical ES. The basal ES is the constructive part of the blood-testis barrier (BTB), while the apical ES facilitates the development and maturation of the spermatid (Liu et al. 2016). After the specific knockout of Atg5 or Atg7 in the mouse Sertoli cells, the apical and basal ES were disrupted, and the cytoskeleton structure was disorganized, resulting in sperm with malformed heads and low motility. Further research revealed that a negative cytoskeleton organization regulator, PDZ and LIM domain 1 (PDLIM1), was degraded by autophagy; therefore, the deficiency of autophagy led to an abnormal accumulation of PDLIM1 and then the disorder of the cytoskeleton structure and ES assembly (Liu et al. 2016).

During spermiogenesis, the haploid spermatids undergo a series of metamorphic changes, including chromatin condensation, centriole remodeling, acrosome and flagella formation, residual body phagocytosis and others to form spermatozoa. The spermatozoa are subsequently released into the tubule lumen in a process called spermiation (Schlatt and Ehmcke 2014; Khire et al. 2016). The acrosome is a membranous-capped organelle that encircles the anterior part of the sperm, and it possesses an internal acid pH and many hydrolyases that are characteristic of lysosomes (Wang H. et al. 2014). However, the acrosome is larger than the lysosome in mammalian cells, and for this reason, researchers hypothesized that the acrosome originated from the modification of the autolysosome. After Atg7 was knocked out in mouse germ cells, the Atg7-mutant mice were almost completely sterile with malformed acrosomes, which was similar to human globozoospermia. Further study suggested that LC3 might function as an intermediary in the fusion and transportation of Golgi apparatus-derived proacrosomal granules. After fusion with acrosomes, LC3 was recycled or degraded, which was analogous to its roles in the induction of autophagy. Thus, ATG7 and the entire autophagy molecular machinery were likely to have participated in acrosome biogenesis and the acrosome reaction (Wang H. et al. 2014). Apart from the acrosome biogenesis defect, follow-up research revealed that the ‘9 + 2’ structure of the sperm flagellum and cytoplasm removal were impaired after the knockout of Atg7 in the mouse germ cells, and these impairments may result from the disrupted F-actin and microtubules (MTs) in the testis. The negative cytoskeleton organization regulator, PDLIM1, accumulated after Atg7 was knocked out; therefore, the disassembled cytoskeleton organization led to abnormal spermatid differentiation (Shang et al. 2016).

F-actin and MTs are two major systems of the cytoskeleton (Pegoraro et al. 2017). Numerous studies have focused on the roles of the cytoskeleton in the formation and transportation of autophagosomes (Kast and Dominguez 2017), whereas little is known concerning the influence of autophagy on the cytoskeleton. Through quantitative mass spectrometry analysis, researchers found that F-actin was disorganized in the autophagy-deficient Atg7^−/- mouse embryonic fibroblast cells (MEFs) (Zhuo et al. 2013). In-depth investigation revealed that a negative regulator of F-actin, PDLIM1 was degraded by autophagy (Liu et al. 2016; Shang et al. 2016). Analogously, another group uncovered that induction of autophagy can stabilize the MTs by degrading superior cervical ganglia protein 10 (SCG10), a MT-disassembly protein in neurons (He et al. 2016). Hence, by degrading negative regulators, autophagy maintains the cytoskeletal organization of certain structures, such as ES and flagella. With new discoveries emerge, autophagy may have more functions in cytoskeletal organization.

After spermiation, the residual spermatozoa in the seminiferous tubules must be degraded in the Chinese soft-shelled turtles, which guarantee the initiation of the next reproductive cycle (Ahmed et al. 2017). During the hibernation period of the Chinese soft-shelled turtles, degraded spermatozoa were enveloped inside the large entotic vacuoles, and different types of autophagosomes could be seen within the entotic vacuoles of Sertoli cells under TEM. The authors suggested that autophagy may be involved in the elimination of germ cells within Sertoli cells (Ahmed et al. 2017).

Autophagy in the testicular endocrinology

Testosterone is an indispensable hormone for male development and maintaining sexual function, and it is mainly synthesized in Leydig cells. The precursor for testosterone biosynthesis is free cholesterol, which is mainly derived in three ways: (1) de novo synthesis from acetate; (2) hydrolysis of stored cholesteryl ester (CE) like lipid droplets (LDs) or the plasma membrane; (3) or obtained from the plasma circulating lipoproteins, such as high-density lipoprotein (HDL) (Azhar et al. 2003). Subsequently, cholesterol is delivered to the mitochondria by transport proteins, such as the steroidogenic acute regulatory (StAR) protein (Stocco 1999). Within the mitochondria, the cholesterol is converted to pregnenolone by cholesterol side chain cleavage enzyme (CYP11A1), then the pregnenolone enters the smooth endoplasmic reticulum and is catalyzed by three steroidogenic enzymes to generate testosterone (Ye et al. 2011). (Figure 2)

Figure 2. The process of testosterone biosynthesis in the Leydig cell. The substrate for testosterone biosynthesis is free cholesterol, which derives from three main sources: (1) de novo synthesis from acetate in the endoplasmic reticulum; (2) hydrolysis of cytoplasmic stored CE (cholesteryl esters), such as LDs (lipid droplets); (3) internalization of plasma circulating lipoprotein, such as LDL (low-density lipoproteins) and HDL (high-density lipoproteins) by respective receptors on the membrane. The LDL-receptor complex is endocytosed into the cell and dissociated by the enzymes of the lysosomes, then the receptor recycles back. In contrast with the low-capacity of the LDL pathway, the bulk uptake of plasma lipoprotein is through the HDL pathway. The CE of the HDL may be stored in the LDs or directly used for testosterone production. Subsequently the cholesterol is transported to the inner mitochondria membrane by specific protein like the StAR (steroidogenic acute regulatory) protein, followed with the catalysis by CYP11A1 (cholesterol side chain cleavage enzyme) to convert into pregnenolone. Finally, the conversion of pregnenolone into testosterone is accomplished in the smooth endoplasmic reticulum.Recent studies revealed that autophagy participates in the hydrolysis of intracellular LDs (①) and facilitation of cholesterol uptake by degrading a negative regulator of HDL receptor (②).

Recent studies have demonstrated that autophagy participates in testosterone production by providing substrates. Scavenger receptor class B, type I (SR-BI), which is recognized as an authentic HDL receptor, promotes the selective uptake of lipoprotein-derived CE (Azhar and Reaven 2002). Recent research demonstrated that a SR-BI-negative regulator, Na+/H+ exchanger regulatory factor 2 (NHERF2), was degraded through the autophagy-lysosome pathway. After knockout of Atg5 or Atg7 in the mouse Leydig cells, the abnormally accumulated NHERF2 downregulated SR-BI, which resulted in deficient cholesterol uptake and finally declined testosterone biosynthesis (Gao et al. 2018). The regulatory function of autophagy towards lipid metabolism, which is also recognized as lipophagy, has been identified in many types of cells, such as hepatocytes, macrophages and adipocytes (Singh and Cuervo 2012). Lipid droplets are sequestered by autophagosomes and then delivered to the lysosomes for degradation to generate free fatty acids (Liu and Czaja 2013). In the Leydig cells, the LDs release free cholesterol (FC) as the substrate for the synthesis of testosterone upon hormone stimulation (Shen et al. 2016). We have observed that after inhibiting autophagy with CQ or siAtg7 in the primary Leydig cells of rats, there is a decline in the level of testosterone and FC, in contrast with an increase in the level of total cholesterol (TC) and LDs in the serum-free medium. Furthermore, short-term hypoxia promoted testosterone secretion and decreased the size and number of LDs, and these changes were negated by blocking autophagy. Thus, autophagy participated in testosterone biosynthesis through the degradation of intracellular LDs/TC (Ma et al. 2018). Similarly, lipophagy was also observed in the Chinese soft-shelled turtle (Tarique et al. 2019).

Of note, other interconnections between testosterone and autophagy may exist. In aged rat Leydig cells, autophagy deficiency synchronized with the low levels of testosterone and StAR protein, which was simultaneously accompanied by the increased dysfunctional mitochondria and ROS levels. After the addition of the autophagy inhibitor wortmannin, there was a decline in the expression of testosterone and luteinizing hormone (LH)-stimulated StAR protein in both young and aged Leydig cells, whereas the autophagy inducer rapamycin, had the opposite effects in aged Leydig cells. Since ROS suppress StAR expression and testosterone synthesis in Leydig cells, therefore it can be proposed that Leydig cell steroidogenesis may be impaired by the accumulation of ROS in aged rats due to reduced autophagic activities (Li et al. 2011).

In nonbreeding males of the natal naked mole-rats (NMRs), a significant decrease in autophagy was associated with decreased testosterone production in the Leydig cells. Treatment with rapamycin increased the testosterone in the breeding and nonbreeding NMR primary Leydig cells, whereas 3-MA exhibited the opposite effects (Yang et al. 2017). These results add weight to the assumption that autophagy may function as a regulator of testosterone biosynthesis; consequently, we assume that the autophagy-inhibition drugs such as rapamycin (CQ) should be cautiously used for patients with reproductive demand, especially those with varicocele or hypogonadism. CQ, a drug used for the initial treatment and prevention of malaria, is widely prescribed in treating rheumatoid arthritis and lupus erythematosus (Rainsford et al. 2015). CQ has been reported to adversely affect male steroid homeostasis and the structural integrity of testes (Okanlawon et al. 1993; Nicola et al. 1997; Clewell et al. 2009); as an autophagy inhibitor, it may negatively influence male fertility through the suppression of autophagy.

ABP is a testicular glycoprotein that is secreted by the Sertoli cells, and it facilitates the transportation of testosterone or dihydrotestosterone into the seminiferous tubules (Della-Maria et al. 2002). In rats, follicle stimulating hormone (FSH) and testosterone appeared to collaboratively promote the transportation of ABP into the epididymis. However, after the administration of FSH, the amount of ABP in the testis increased by only one-sixth of that when testosterone was administrated; hence, testosterone may be the dominant regulator of ABP synthesis in vivo (Danzo et al. 1990). A detailed study demonstrated that in rat Sertoli cells, testosterone seemed to selectively manipulate the autophagic degradation of ABP, both in vitro and in vivo, since the expression of ABP was not changed by CQ or rapamycin after the administration of testosterone. Moreover, the fact that the clearance of ABP was irrelevant to stress (hypoxia)-induced autophagy also adds weight to the assumption that testosterone may be a specific switch controller in the degradation of ABP by autophagy (Ma et al. 2015).

Autophagy contributes to testosterone production by offering materials (Gao et al. 2018; Ma et al. 2018); however, testosterone inhibits autophagy in turn (Ma et al. 2015). Therefore, we speculate that testosterone may function as a negative feedback loop towards autophagy to sustain cellular homeostasis. Through participating in testosterone production and ABP metabolism, autophagy may indirectly influence on the process of spermatogenesis.

The role of autophagy in the existence of environmental endocrine-disrupting chemicals

Autophagy can be induced or enhanced by various external factors, such as hypoxia and endocrine-disrupting chemicals (EDCs). These stimuli could influence the spermatogenic or endocrinological procedure through the autophagic pathway. Among these, EDCs have been extensively focused.

EDCs, referred to as environmental substances that can interfere with the endocrine functions in the body and thus exert harmful effects on the organism or their offspring, are ubiquitous in our surroundings (Knez 2013). EDCs can impair male reproductive health by disturbing the production, secretion, transport, binding and excretion of endogenous hormones or the spermatogenesis process through direct and indirect methods (Knez 2013; Den Hond et al. 2015). Autophagy is a double-edged sword when the organism is confronted with external insults. To a certain extent, it serves as a survival strategy to surmount a myriad of barriers, but excessive autophagy could also promote cell death.

Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin, can be found in many food products. In ZEA-treated rat Leydig cells, the levels of apoptosis and autophagy are upregulated. Pretreatment with the autophagy inhibitor CQ increased the rate of apoptosis, while rapamycin decreased the rate of apoptosis, which implied a protective role of autophagy against ZEA-induced apoptosis (Wang Y. et al. 2014). Similar results can be concluded from the ZEA-treated rat Sertoli cells, xenoestrogen 4-nonylphenol (NP)-treated rat Sertoli cells, bisphenol A (BPA)-treated goat Sertoli cells, and dibutyl phthalate (DBP)-treated prepubertal rat germ cells (Duan et al. 2016; Zhang G. et al. 2016, Zhang Y. et al. 2017; Wang et al. 2018). In contrast, after being treated with a high dose of microcystin-LR (MC-LR), the accumulated autophagosomes seemed to facilitate the apoptotic process by elevating cytochrome C (Cyt C) and caspase 3 in rat Sertoli cells. In addition, the MC-LR-induced abnormalities were alleviated after the treatment with the autophagy inhibitor 3-MA (Chen et al. 2013). A cytotoxic role of autophagy could also be observed in the di-2-ethylhexyl phthalate (DEHP)-treated mouse TM3 Leydig cells (Sun et al. 2018).

External stimuli-induced autophagy is mostly selective, which may be different from the basal autophagy in the spermatogenesis and testicular endocrinology. This phenomenon can be demonstrated by the research study that found that the degradation of ABP was independent of hypoxia-induced autophagy (Ma et al. 2015). Accordingly, the mechanisms of stimuli-induced autophagy deserve further exploration.

Conclusion

Cumulative achievements revealed that autophagy is active in many aspects of male spermatogenic and endocrinological processes, nevertheless, this is only the tip of the iceberg. By facilitating cholesterol uptake and degrading intracellular LDs for testosterone biosynthesis, autophagy may be involved in the metabolism and elimination of testosterone, as well as in the production of other hormones that use cholesterol as substrates. A full-scale exposure of autophagy would expand our understanding of the male reproductive system, which may provide novel therapeutic strategies for male infertility.

Authors’ contributions

Contributing to the writing of the manuscript and drawing of review figures and table: YZ; Carried out the collection of relevant data and literature: QY, DW, ZY; Conceived the draft and participated in modification: YD and YM. All authors have read and approved the final version of the manuscript, and agree with the order of presentation of the authors.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China under Grant (No.81501310 and No.81871199) and Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University under Grant (RJZZ14-009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.