Environmental oestrogens disrupt testicular descent and damage male reproductive health: Mechanistic insight

Abstract Environmental oestrogens (EEs) as environmental pollutants have been paid much attention due to their impact on congenital malformation of male genitourinary system. Exposure to EEs for prolonged time could hinder testicular descent and cause testicular dysgenesis syndrome. Therefore, it is urgent to understand the mechanisms by which EEs exposure disrupt testicular descent. In this review, we summarize recent advances in our understanding of the process of testicular descent, which is regulated by intricate cellular and molecular networks. Increasing numbers of the components of these networks such as CSL and INSL3 are being identified, highlighting that testicular descent is a highly orchestrated process that is essential to human reproduction and survival. The exposure to EEs would lead to the imbalanced regulation of the networks and cause testicular dysgenesis syndrome such as cryptorchidism, hypospadias, hypogonadism, poor semen quality and testicular cancer. Fortunately, the identification of the components of these networks provides us the opportunity to prevent and treat EEs induced male reproductive dysfunction. The pathways that play an important role in the regulation of testicular descent are promising targets for the treatment of testicular dysgenesis syndrome.


| INTRODUC TI ON
Congenital malformation of male reproductive tract is one of the most common malformations at the birth. 1 With the development of industry and environmental pollution, the incidence of congenital malformation of genitourinary system is increasing.
Environmental oestrogens (EEs) as environmental pollutants have been paid much attention due to their impact on congenital malformation of male genitourinary system. EEs are external chemical pollutions caused by human activities such as industry and agriculture, and can be categorized into four groups: naturally occurring non-steroidal plant oestrogens or phytoestrogens; steroid oestrogens-17β oestradiol and oestrone from animal and human sources; mycotoxins, zearalenone and zearalenol; synthetic compounds with phenolic groups. 2 EEs are mainly found in pesticides, food additives, plastics and they are stable in the environment, such as sewage water used for irrigation at concentrations that could affect lucerne growth. The dietary contribution of oestrogenic industrial compounds is 0.0000025% of the daily intake of oestrogenic flavonoids in the diet. 3 by intricate cellular and molecular networks. Increasing numbers of the components of these networks such as CSL and INSL3 are being identified, highlighting that testicular descent is a highly orchestrated process that is essential to human reproduction and survival. The exposure to EEs would lead to the imbalanced regulation of the networks and cause testicular dysgenesis syndrome such as cryptorchidism, hypospadias, hypogonadism, poor semen quality and testicular cancer. Fortunately, the identification of the components of these networks provides us the opportunity to prevent and treat EEs induced male reproductive dysfunction. The pathways that play an important role in the regulation of testicular descent are promising targets for the treatment of testicular dysgenesis syndrome.

K E Y W O R D S
androgen, environmental oestrogen, infertility, INSL3, testicular dysgenesis syndrome, undescended testes It is reported that the incidence rates of testicular germ cell tumours, low semen quality, cryptorchidism and hypospadias in male are related to prenatal environmental chemical exposure based on human and animal studies. 4 In particular, EEs have been proved to be closely related to undescended testes (UDT) not only in epidemiological investigation, but also in many experimental studies on human and animals (Table 1). [5][6][7][8][9][10][11][12][13][14] EEs lead to a series of developmental malformations of male urogenital system due to testicular insufficiency, among which UDT is one important manifestation. Diseases related to UDT include but are not limited to cryptorchidism, sexual dysfunction, testicular atrophy, infertility and testicular tumours.
Similar to oestrogen, EEs exert biological effects by binding to oestrogen receptor (ER) to regulate gene transcription in the nucleus or act in a non-genetic manner to activate intracellular signalling pathways such as calcium and kinases in the cytoplasm to regulate cytoskeleton. 8,9,14 At present, the mechanism by which EEs affect testicular descent is not completely understood.
Therefore, in this review we aimed to summarize recent advances in our understanding of the role of EEs in the process of testicular descent, propose the mechanisms by which EEs affect testicular descent and highlight potential targets to block the vicious effects of EEs on genitourinary system in order to improve human health.

| DE VELOPMENT OF TE S TICUL AR DE SCENT
Testicular descent is a development process including two divided phases, transabdominal and inguinoscrotal phase ( Figure 1). First, at transabdominal phase, testis originate from urogenital ridges, and begin to move to the inguinal ring at 10-15th week in human embryonic period. 15 In the early foetus, the gubernaculum is seen as a short, thin ligament connecting the ambisexual gonad and the urogenital ridge containing Wolffian ducts (WDs) to the inguinal region. 15 The degeneration of cranial suspensory ligament (CSL) at the top of male foetal urogenital crest, coupled with the expansion of the enlargement of the gubernaculum through mitosis and deposition of hyaluronic acid, eventually brings the testes very close to the future inner inguinal ring. This stage mainly depends on the interaction between insulin-like factor 3 (INSL3) and its receptor. 16 Testosterone is thought to be the sole factor responsible for the stabilisation of the WDs. Leydig cells produce testosterone at 8 weeks of gestation in humans, and testosterone is thought to be secreted directly into the WDs by diffusion. 16 Meanwhile, androgen stimulates suspensory ligament degrade. This action will reduce the counterforce for testicular descent. The WDs then develop into separate but contiguous organs, the epididymis, vas deferens and seminal vesicles. 16 In the inguinoscrotal phase, the gubernaculum migrates and elongates towards the scrotum, and eventually the testis descend to the scrotum. Then, at inguinoscrotal phase, testis enter into inguinal ring through groin tube, and reach scrotums. At this phase, testis experience a series of changes. For example, testis, gubernaculum and inguinal ring enlarge, spermatic veins increase and extend.
This step is critically dependent on the function of testosterone. 17

| CON S EQUEN CE OF UDT BY TE S TICUL AR UNDE SCENT
Skakkebaek et al. proposed that undescended testis, hypospadias, poor semen quality and testicular cancer are the manifestations of testicular dysgenesis syndrome, named by others as 'the developmentally estrogenized male phenotype'. 2,18 EEs also have the impact on wild animals, including cryptorchidism of Florida leopard, penile band of male otter, penile fish of alligator, sex reversal of fish and the changes in mating behaviour of birds. [19][20][21] In sensitive pregnancy stage, the exposure to toxic dose of EEs would damage reproductive system, leading to testicular dysgenesis syndrome (TDS) and genitalia malformations. UDT is not only the insufficiency of testicular descent, but also directly or indirectly affects the structural and functional abnormalities of male genitourinary system, including cryptorchidism, hypospadias, testicular cancer and TDS. The declining reproductive ability and testicular tumours are two aspects that we will discuss below.

| UDT DAMAG E S THE FERTILIT Y IN MEN
In the second half of the 20th century, with the development of industrialisation, people began to realize the impact of EEs on male reproductive system. Initial studies showed that the male infertility rate in developed countries increased by 0.5%-1% every year, the proportion of cryptorchidism and hypospadias increased, and the quantity and quality of semen in industrialized countries declined. 2 The direct observations on the human impact of oestrogen come from occupational exposure events, such as the decrease of sperm number, loss of sexual desire and impotence among workers dealing with DDT, Kepone and manufacturing DAS. 22,23 A study by American Society for Reproductive Medicine in 2014 on 149 infertile men showed that bisphenol A concentration in urine and sperm motility was negatively correlated. 24 In addition, investigation of infertile men showed that 10% infertile men had a UDT history. Double UDT patients had six times high probability than single UDT patients. 25 It was reported that the probability of azoospermia in single UDT patients was 13.3% and increased to 88.6% in double UDT patients.
The probability of azoospermia in double UDT patients fell to 46% and 32% after treated with orchiopexy and hormone, respectively, while no significant change in single UDT patients. 26 These results provide strong evidence that UDT damages the fertility in men.

| UDT C AUS E S TE S TI CUL AR TUMORIG ENE S IS
Testicular cancer is the most common malignancy among men between 14 and 44 years old, and its incidence has risen over the past two decades in Western countries. 27 Both genetic and environmental factors contribute to the occurrence of testicular cancer. 28     U-73122 inhibited diethylstilbestrol-induced cAMP-response element binding protein activation in gubernaculum testis cells TA B L E 1 (Continued) exposed to hormone disruptors had higher incidence of testicular cancer and seminoma. 29 Cryptorchidism is one of the most common congenital abnormalities in boys, and is one of the few known risk factors for testicular cancer. 30 In patients with cryptorchidism, testis development is abnormal because the testis is not located in the scrotum. The temperature of other places is higher than that in scrotum. Therefore, the risk of testicular tumour is 35-48 times higher in cryptorchidism patients than in general population. In addition, 3%-18% of cryptorchidism patients will develop testicular cancer. 31 It was found that elevated concentrations of polychlorinated biphenyls, hexachlorobenzene and chlordanes were detected in the blood of mothers of men with testicular cancer. 32 There is a positive correlation between household use of pesticides, especially fungicides, in early development and the risk of adult testicular germ cell tumours and non-seminoma. 33

| THE MECHANIS MS BY WHI CH EE s D IS RUP T TE S TI CUL AR DE SCENT
EEs do not cause instantaneous toxicity because of low concentration in the body. However, EEs can go through the blood-placenta barrier to affect foetal development on maternal pregnancy. 34 EEs are harmful to male genitourinary system especially during pregnant stage. EEs could hinder testicular differentiation and descent during embryonic stage. In the entire testicular descent process, many factors are involved to regulate testicular descent. EEs can interference with one or more of these factors to hinder testicular descent. 35

| IMPAC T OF EE s AT TR ANSAB DOMINAL PHA S E
As shown in Figure 1, androgen receptors, oestrogen receptors (AR and ER) and the G protein-coupled oestrogen receptors (GPER). DES could impede gubernaculum differentiation through ERK1/2 signalling pathway. 8,9 Therefore, EEs could impair the process of testicular descent by inhibiting the development of gubernaculum during pregnancy.

| IMPAC T OF EE S AT ING U INOSCROTAL PHA S E
In androgen insensitivity syndrome patients and in animals with anti-androgen treatment, testicular descent failed in inguinoscrotal stage. 43,44 From endocrinology perspective, testicular Leydig cell se- CGRP then binds to the receptors on the gubernaculum, and the gubernaculum shrink and lead testicular into scrotum to complete the process of testicular descent.

| IMPAC T OF EE S ON TE S TOS TERONE ME TABOLIS M
Testosterone (T) is the most common androgen. The expression of P450 17α hydroxylase in testicular Leydig cell of male foetal rats decreased after the female rats were treated by EEs during pregnancy. 46 Therefore, EEs could inhibit cytopigments P450 (CYP450) to reduce the production of T. Phthalates increase the production of testicular testosterone in foetal rats (low-dose effect), but high dose will reduce the production of testicular testosterone in foetal rats, resulting in a lower incidence of anogenital distance and increased incidence of cryptorchidism. 47 Some studies found that EEs could suppress 5α-NADPH activation in the metabolism of T. In addition, T converts to oestra-

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.