Public concern regarding environmental contaminants (ECs)-related reproductive disorders has increased due to increasing global rates of infertility. All kinds of ECs are on rise rapidly in developing and industrializing low- and middle-income countries. The aquatic environments throughout the world are repositories for enormous amounts of ECs. As the biology of the reproductive system is highly conserved in vertebrates, wildlife or laboratory studies on fish provide significant information to establish a detailed risk assessment, and to identify novel or more sensitive endpoints for ECs-related reproductive disorders. The adverse effects of ECs on endocrine regulation of reproduction in male fishes have been extensively studied and reviewed; however, our knowledge on the effects and mechanisms of action of ECs on determinants of male fertility is limited.
Global rates of environmental contaminants (ECs)-related reproductive disorders have been increasing over the past 50 years. In human beings, the incidences of testicular dysgenesis syndrome, including hypospadias (urethra opens on the underside of the penis instead of the tip), cryptorchidism (one or both testes not descended into the scrotum), testicular cancer, low semen quality, and infertile men, show global increases associated with ECs 
. Landrigan et al. 
reported that all kinds of ECs are all on the rise rapidly in developing and industrializing low-income and middle-income countries. The public concern regarding ECs-related reproductive disorders was originally linked to observations of reduced fertility, birth defects, and sexual developmental disorders in wildlife 
. For over 30 years, the World Health Organization (WHO), National Institute of Health (NIH, USA), European Food Safety Authority, and the other organizations composed of working groups of experts in endocrinology, risk assessment, and toxicology, have conducted studies to examine the adverse effects of ECs on reproduction in humans and wildlife. These studies have shown that there are about 800 natural and man-made chemicals known or suspected to interfere with physiological and endocrinological regulation of reproduction 
. However, our knowledge on the ECs-related hormonal dysfunctions that cause diminished fertility is limited to a small fraction of these chemicals. To reduce ECs-related fertility threat in males, it is critical to identify the contaminants that interfere with determinants of fertility, including sperm production, morphology, genome, and motility, and to characterize their modes of action on reproductive endocrine system. In this regard, interdisciplinary efforts that combine knowledge from wildlife, experimental animals, and human infertility clinics are needed to provide a more holistic approach for ECs-related reproductive disorders and fertility threat.
The aquatic environment is at greatest risk from pollutants since all chemicals will eventually find themselves in the rivers, lakes, and oceans as the final repository 
. As biology of reproduction is highly conserved in vertebrates 
, studies on fishes as model organisms provide significant information to establish a detailed risk assessment and to establish novel or more sensitive endpoints for ECs-related fertility threat. Frequent clear evidences show reproductive disorders in fishes from polluted aquatic environments (see Section 4
). The adverse effects of ECs on endocrine regulation of reproduction in male fishes have been extensively studied and reviewed in laboratory studies 
. In contrast, our knowledge to understand whether ECs-disrupted hormonal functions result in diminished fertility is poor. To answer, it is critical to uncover the adverse effects of ECs on sperm production, morphology, genome, and motility kinetics as key determinants of fertility.
We have recently reviewed the toxicity of ECs on sperm morphology and motility in fishes, in vitro 
. The review showed that ECs, in a dose-dependent manner, cause damage to sperm morphology and interfere with sperm energetics and motility kinetics, and thus affect male fertility. However, significant decreases or complete suppression of sperm motility and fertilizing ability occurred mostly at concentrations considerably higher than those reported in the aquatic environment or exceeding the WHO recommended limits for surface waters. Recently, Carnevali et al. 
and Golshan and Alavi 
suggested that ECs are capable of affecting sperm quality in fishes associated with alternations in hormonal functions of hypothalamus–pituitary–testis (HPT).
2. An Introduction to Reproductive Biology in Male Fishes
It is essential to review reproductive biology, fertility indicators, and determinants of fertility in male fishes before delving into the ECs-related fertility threat. These provide the basic information to better understand multiplicity of sites through which ECs interfere with fertility. To clarify the terminology, “semen” refers to seminal plasma and sperm and “sperm” refers to sperm cells in the present review.
2.1. Anatomy of Reproductive Organ
In general, the male reproductive organ consists of a paired testes, the testicular duct, and the sperm duct in fishes 
). In some primitive fishes (such as sturgeons), the testes release sperm into the testicular ducts, which pass the kidneys. At spawning, semen is released into the aquatic environment through the urinary ducts opened into the urogenital opening (Figure 1
A,C). In most bony fishes (teleosts), neither testicular ducts nor sperm ducts attach to the kidneys. The sperm is released from the testes into sperm ducts where seminal plasma is secreted. At spawning, semen is released into the aquatic environment through the sperm ducts opened in the urogenital opening (Figure 1
Reproductive system in male fishes. Panels (A
) show the anatomy of the reproductive system in primitive fishes (sturgeons). Sperm is released from the testes into the testicular ducts, which pass the kidney. At spawning, semen is released into the aquatic environment through the urinary ducts opened into the urogenital opening (UO). Panels (B
) show the anatomy of the reproductive system in bony fishes (teleosts). Sperm is released from the testes into the testicular ducts. At spawning, semen is released into the aquatic environment through the sperm ducts opened into the UO. Panels (E
) are schematic of the tubular testis and lobular testis, respectively. Panel G shows testicular compartments in fishes. K, kidneys; SD, sperm duct; SC-I, primary spermatocyte, SC-II, secondary spermatocyte; SG, spermatogonia; SP, spermatid; SZ, sperm; T, testis; TD, testicular duct; UB, Urinary bladder; UD (WD), urinary duct (Wolffian duct). The panels are modified from Grier 
, Nagahama 
; Alavi et al. 
and Dzyuba et al. 
. The photo of panel A is courtesy of Associate Professor Borys Dzyuba from the sterlet (Acipenser ruthenus
). The photo of panel B is from S. M. H. Alavi from the Northern pike (Esox Lucius
). Panels C-G credits: © S. Barzegar-Fallah.
The testes are divided into the “tubular type” and the “lobular type” according to the distributions of spermatogonia in the seminiferous region 
In the tubular type, as spermatogonia divide and enter in meiosis, the cysts migrate towards the region of the spermatic ducts located in the central region of the testis, where the cysts open to release sperm (Figure 1E). This type of testicular arrangement is found in zebrafish (Danio rerio) and guppy (Poecilia reticulate).
In the lobular type, the testis is composed of numerous lobules that are separated from each other by a thin layer of fibrous connective tissue, and spermatogonia are spread along the germinal compartment throughout the testis. The cysts do not migrate or become displaced during their development, and sperm is released into the lobular lumen (Figure 1F). This type of testicular arrangement is found in Japanese medaka (Oryzias latipes), common carp (Cyprinus carpio), goldfish (Carassius auratus), and rainbow trout (Oncorhynchus mykiss).
The testicular compartment contains Sertoli cells, Leydig cells, blood/lymphatic vessels, macrophages and mast cells, and neural and connective tissue cells (Figure 1G). The Leydig and Sertoli cells are involved in biosynthesis of steroid hormones to regulate sperm production and maturation.
The testicular ducts are located adjacent to the testes, which continue into the sperm ducts on the ventral sides. Testicular and sperm ducts possess very similar structural and enzyme-histochemical characteristics, and play key roles in nutrition of sperm, storage of sperm, synthesis of steroids, secretion of proteins and enzymes, and formation of the seminal plasma 
. Maturation of sperm to acquire potential for motility and fertilizing ability occurs in the sperm ducts 
Sperm is produced from spermatogonia following divisions 
. During the process of spermatogenesis, diploid spermatogonia type A divides mitotically to produce diploid spermatogonia type B. The final mitotic division of spermatogonia type B produces diploid primary spermatocytes that undergo the first meiotic division to form haploid secondary spermatocytes. The second meiotic division produces haploid spermatids that transform into the flagellated sperm.
2.3. Sperm Morphology
Sperm is differentiated into a head, midpiece, and flagellum in fishes 
). The head of sperm contains DNA for transferring a haploid set of the chromosomes into the oocyte upon fertilization. Mitochondria and proximal and distal centrioles are located in the midpiece. Mitochondria deliver energy that is required for the beating of the sperm motility apparatus with a “9 + 2” structure called the “axoneme” 
. Both proximal and distal centrioles consist of nine peripheral triplets of microtubules. The distal centriole forms the basal body of the axoneme. Sperm is acrosomeless in teleostean fishes, while it possesses acrosome in primitive fishes, including hagfish and sturgeons 
Sperm morphology in primitive (chondrostei) and bony (teleostei) fishes. Sperm is composed of a head (nucleus, N), midpiece (M) and flagellum (F). In chondrostei fishes (such as sturgeons), there is an acrosome (A) at the top of the head of sperm. The ultrastructure compartments of sperm are similar between chondrostei and teleostei fishes: DC, distal centriole; PC, proximal centriole; Mt, mitochondria. The structure of the motility apparatus called “axoneme” is highly conserved, and possesses the typical 9 + 2 microtubule structure of cilia surrounded by plasma membrane. The electron micrographs are selected from the Russian sturgeon (Acipenser gueldenstaedtii
, and Atlantic halibut (Hippoglossus hippoglossus
) sperm 
. The schematic of the axoneme is from Inaba 
2.4. Sperm Physiology
The seminal plasma is a product of Sertoli cells, testicular ducts, and sperm ducts, and its composition is different among fishes that may reflect species variations. The main role of seminal plasma is to create an optimal environment for the storage of sperm during maturation in the sperm ducts. Seminal plasma maintains sperm viability, motility, and fertilizing ability, and protects sperm against damage caused by proteolytic or oxidative attacks 
2.5. Sperm Motility
Sperm is generally immotile in the seminal plasma and the sperm ducts of fishes, and motility is triggered upon discharge into the aquatic environment (Figure 3
A). In most freshwater and marine fishes, osmolality of the seminal plasma is the key factor to maintain sperm in the quiescent state in the sperm ducts 
. In some freshwater fishes, including Salmonidae and Acipenseridae, high concentrations of potassium (K+) ions inhibits sperm motility in the seminal plasma 
. At spawning, a hypo-osmotic and a hyper-osmotic signal is necessary for initiation of sperm motility in freshwater and marine fishes, respectively 
. Changes of osmolality around sperm accompanied by K+ efflux in freshwater fishes and water efflux in marine fishes trigger sperm motility signaling. Activation of sperm motility is associated with an increase in intracellular pH and calcium (Ca2+
) ions in both freshwater and marine fishes, while cyclic adenosine monophosphate (cAMP) remains unchanged. However, studies show that demembranated sperm of salmonid and sturgeon fishes require cAMP for the axonemal beating 
. In some marine fishes, it has been shown that 17,20β
-P) is capable to induce sperm hypermotility by increasing cAMP and intracellular Ca2+
through a membrane progesterone receptor 
Figure 3. Sperm motility signaling and kinetics in fishes. Panel (A) summarizes sperm motility signaling in fishes. Sperm is immotile in the sperm ducts and seminal plasma. At spawning, a hypo- osmolality accompanied by K+ efflux or hyper-osmolality accompanied by water efflux trigger sperm motility activation in freshwater and marine fish species, respectively. Activation of ATP- dependent sperm motility initiation is associated with an increase in intracellular calcium ([Ca2+]i) ions in all fish species and an increase in intracellular potassium ([K+]i) ions in marine species, while cyclic adenosine monophosphate (cAMP) remains unchanged. However, demembranated sperm of salmonids requires cAMP for axonemal beating. Panel (B) shows sperm motility kinetics in fishes. After initiation of sperm motility, percentage of motile sperm and sperm velocity decrease rapidly in both freshwater and marine fishes due to depletion of adenosine triphosphate (ATP) content. Panel (C) is a schematic representing various sperm velocity parameters analyzed by a computer-assisted sperm analysis. The curvilinear velocity (VCL) is the velocity along the trajectory of sperm head. The straight line velocity (VSL) is the straight line distance between the start and end points of the track divided by the duration of the movement. The angular path velocity (VAP) is the velocity along a derived smoothed path.
After initiation of sperm motility in the aquatic environment, duration of motility is very short in fishes from a few seconds to several minutes or hours depending on the species 
. The inter-species differences probably depend on the capacity of the sperm to restore intracellular ATP and creatine phosphate concentrations 
. Once sperm motility is initiated, the percentage of motile sperm and sperm velocity rapidly decrease, which are associated with a large, but not complete depletion of ATP 
B). Fish sperm can regenerate ATP from phosphocreatine and ADP; however, this ATP regeneration system does not prevent the precipitous decline in ATP levels during motility 
3. Fertility Indicators and Assessments in Fishes
Fertilization and hatching rates are calculated to assess fertility in fishes (Figure 4
A). The fertilization rate is the percentage of oocytes that become fertilized upon spawning or artificial insemination, and is calculated as number of fertilized eggs/initial number of oocytes × 100. Successful fertilization depends on onset of release of sperm from males and ova from females 
. During the short period of motility, sperm must penetrate the oocyte through a funnel called the “micropyle” to fertilize it 
. A fertilized egg can be easily identified by the presence of a multi-cellular blastodisc (cleavage), which occurs from several hours to a few days post fertilization and depends on fish species and environmental factors including temperature. The hatching rate is the percentage of hatched larvae, and calculated as number of hatched larvae/initial number of oocytes × 100. Once embryonic development is completed, larvae hatch 
Figure 4. Indicators and determinants of fertility in male fishes. Fertilization success is assessed by fertilization rate or hatching rate (A). Sperm production, morphology, genome and motility are key determinants of fertility in male fishes (B).
4. Determinants of Fertility in Male Fishes
Analyses of sperm production, morphology, genome, and motility kinetics are basically important to assess fertility in male fishes (Figure 4B).
4.1. Sperm Production
Frequent studies have shown that fertilization rate positively correlates with sperm volume, sperm density, number of sperm per oocyte, and density of sperm in the water during fertilization 
. One can weigh semen mass, measure semen volume, or count sperm density to evaluate sperm production.
4.2. Sperm Morphology
There is a species-specific relationship between the head size of sperm and diameter of micropyle in fishes 
. This indicates that sperm of one species can penetrate only into the oocyte of similar species. A change in the size of sperm head is a mirror of the size of nucleus 
. It has also reported that sperm with a smaller head can move faster than those with a larger head 
. Additionally, both positive and negative correlations have been reported between the length of flagellum and the sperm velocity 
. These suggest that alternations in the size of sperm head and length of flagellum can result in diminished fertility by affecting sperm penetration into the oocytes or sperm motility performance. Various microscopic techniques including scanning and electron microscopy are valuable methods to assess sperm morphology 
4.3. Sperm Genome
Upon fertilization, sperm with a haploid number of chromosomes transmit a parental genome to the next generation. Alternation of chromosome material, Y chromosome deletion, and ploidy level are among factors that affect fertility. The integrity of sperm DNA correlates with fertilization and embryonic development in fishes 
. Fertility threat has been frequently reported in polyploid fish, which were associated with failure of testicular development 
, enlarged head size making penetration of sperm through a normal-sized micropyle difficult 
, reduced sperm production 
, and increased abnormal sperm with malformation of the head, mitochondria, and flagellum resulting in decreasing motility and velocity 
. One can assess the integrity of DNA using a comet assay, sperm chromatin structure assay, or terminal deoxynucleotidyl transferase dUTP nick end labelling assay (TUNEL) 
. Chromosome number and DNA content can be counted or assessed using a flow cytometry, respectively 
4.4. Sperm Motility Kinetics
Duration of sperm motility, percentage of motile sperm, and sperm velocity are key determinants for fertility in male fishes 
. It has been shown that sperm with faster movement and a longer period of motility have more chance to approach an oocyte to fertilize it 
. In addition, it has been suggested that sperm velocity and the duration of motility are positively correlated with ATP content of sperm 
. A computer-assisted sperm analysis (CASA) provides a valuable tool to assess sperm motility kinetics 
. Percentage of sperm motility is evaluated by counting the number of motile sperm and total number of sperm. The sperm velocity is the distance between the starting and ending points of the motility track divided by the time spent for this movement. Based on sperm head positions during the period of motility, various sperm velocity parameters are identified, including curvilinear velocity (VCL, the velocity along the sperm head trajectory), straight line velocity (VSL, the straight line distance between the start and end points of the sperm head trajectory), and the angular path velocity (VAP, the velocity along a derived smoothed path) (Figure 3