REPRODUCTIONREVIEW

Insulin-like factor 3 as a monitor of endocrine disruption

Ravinder Anand-Ivell1,2 and Richard Ivell3,4 1School of Biosciences, University of Nottingham, Sutton Bonington, UK, 2School of Pharmacy and Medical Science, University of South Australia, Adelaide, South Australia 5000, Australia, 3Leibniz Institute for Farm Animal Biology, 18196 Dummerstorf, Germany and 4School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia Correspondence should be addressed to R Anand-Ivell at School of Biosciences, University of Nottingham; Email: [email protected]

Abstract

Insulin-like factor 3 (INSL3) is generated and secreted by differentiated interstitial Leydig cells of the testes in both fetal and adult males of all mammalian species so far analyzed. All evidence to date suggests that it is produced constitutively, independently of acute regulation by the hypothalamo-pituitary–gonadal (HPG) axis, in amounts which reflect the numbers and differentiation status of the Leydig cells. This Leydig cell functional capacity is otherwise monitored only by androgen output, which, however, is massively confounded by acute regulation from the HPG axis and other factors leading to substantial and irregular short-term variation. Leydig cells are a primary target of endocrine-disrupting agents in the context of the testicular dysgenesis syndrome in the fetal male, as well as in the adult. In the male fetus, INSL3 is responsible for the first phase of testicular descent, and hence is directly linked to the etiology of . In this study, by measuring INSL3 production, for example, during fetal life via amniotic fluid, or as secretions from fetal testis explants, or in adult peripheral blood, we and others have shown that INSL3 represents a useful quantitative and sensitive endpoint for assessing the impact of endocrine-disrupting agents and their mechanisms of action. Reproduction (2014) 147 R87–R95

Introduction fetus is indeed vulnerable to a range of intrinsic (maternal nutrition and metabolism) and extrinsic Until relatively recently, the growing embryo in agents, mostly man-made chemicals which appear to viviparous mammals was considered to be protected be able to interfere with the establishment of fetal organ from external influences by a combination of the fetal systems and their endocrine or paracrine control membranes and the placenta, together with the catabolic mechanisms (Swanson et al.2009, Barouki et al. capacity of the maternal physiology, where the liver and 2012). The DOHaD concept is reinforced by our modern kidney act as first lines of defense against noxious understanding of the important role played by epige- environmental agents. Since the construction of the netics, not only in determining cell fates and lineage DOHaD hypothesis (Developmental Origins of Health phenotypes in the embryo, but also by acting as a and Disease), we now know that in spite of these memory for extrinsic insults, which may impact on adult maternal and fetal defense mechanisms the growing physiology long after the exposure has ceased (Sinclair et al. 2007). One of the most vulnerable periods during the life of This paper forms part of a special issue of Reproduction on Endocrine the fetus is in the transition from first to second trimester. Disrupters. This article was presented at the 7th Copenhagen Workshop This is the time when most organ systems are developing on Endocrine Disrupters, 28–31 May 2013. The meeting was supported by the Danish Ministry of the Environment – Environmental Protection rapidly, both anatomically as well as in the establishment Agency as an activity under the Danish Centre on Endocrine Disrupters. of their endocrine control systems. It is also a time Publication of this special issue has been supported by the Society for when the fetal skin is still relatively permeable, such Reproduction and Fertility. The opinions or views expressed in this that amniotic fluid largely comprises fetal serum exudate special issue are those of the authors, and do not necessarily reflect (Underwood et al. 2005, Beall et al. 2007), and thus the opinions or recommendations of the Danish Ministry of the offers very little protection against exogenous chemical Environment – Environmental Protection Agency or the Society for insult. Moreover, at this time the placenta is establishing Reproduction and Fertility. The Guest Editors for this special issue were Anna-Maria Andersson, Hanne Frederiksen, Niels Erik Skakkebæk, its vascular bed and functional dimensions. Typical for Rigshospitalet, Denmark, Kenneth M Grigor, Western General Hospital, this period is the differentiation of the male organs Edinburgh, UK and Jorma Toppari, University of Turku, Finland. of reproduction, the testes and Wolffian derivatives.

q 2014 Society for Reproduction and Fertility DOI: 10.1530/REP-13-0486 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/28/2021 11:36:45PM via free access R88 R Anand-Ivell and R Ivell

Very shortly after SRY-induced sex determination in the expression of the SRY by the Sertoli cells to signal male fetus, the somatic cells of the fetal testes proliferate sex determination. In humans, this occurs at around and differentiate into Sertoli and steroidogenic Leydig week 7 of pregnancy and in mice at around day 11 (Viger cells, providing both a niche for the developing et al. 2005, Sekido & Lovell-Badge 2013). Latest totipotent germ cell stem cells (gonocytes) as well as evidence suggests that the fetal Leydig cells (at least in delivering the essential for adequate develop- the mouse) may have a dual origin, in that they appear ment of the male genitalia and reproductive organs not only to differentiate from mesenchymal cells within (androgens, AMH, and insulin-like factor 3 (INSL3)). the gonadal ridge but also migrate in from close to the In contrast, the ovary of the female fetus at this time is mesonephros (De Falco et al. 2011). In either case they relatively less active as an endocrine organ, with a lack of begin to express the transcription factor SF1 almost hormones determining the female phenotype. Therefore, immediately after Sry expression in the Sertoli cells at because the testes are very actively differentiating at this around day e10.5 in the mouse (Sekido & Lovell-Badge vulnerable time, and because they are both producing 2008). INSL3 is a gene which in the adult testis is and are themselves regulated by a very active hormonal regulated via multiple SF1-responsive elements in its system in this period of early gestation, environmental promoter, but is a marker for a relatively late stage of endocrine disruption by extrinsic -like factors Leydig cell differentiation (Sadeghian et al. 2005). If its has been most emphatically observed in regard to the expression is similarly regulated in the fetus, then this male reproductive phenotype. Consequently, symptoms would imply that INSL3 in fetal Leydig cells should of the testicular dysgenesis syndrome (TDS; i.e. cryp- become detectable soonest after about day e12.5 in the torchidism, hypospadias, testis cancer, reduced sperm mouse, or by weeks 8–10 in the human. Indeed, the earliest counts, and adult androgen production) are among the evidence for Insl3 mRNA detection in the mouse is on commonest manifestations of endocrine disruption day e12.5 (Sarraj et al.2010) and, for the human, INSL3 during pregnancy (Skakkebaek et al. 2001, Sharpe & has been measured in the amniotic fluid Skakkebaek 2008). Nevertheless, other organ systems are at gestational week 11 (R Anand-Ivell, unpublished also likely to be affected, though possibly less obviously. observations). There is less evidence available for other Unfortunately, the transition from first to second species, although sporadic measurements of INSL3 in the trimester of pregnancy in humans is one of the most bovine (Anand-Ivell et al.2011), porcine, or rat systems difficult physiological time points to access. The fetus is (Anand-Ivell, Vernunft, Barthol & Ivell, unpublished still very small and fragile, and maternal serum offers few observations) confirm that high levels of INSL3 are clues as to how the pregnancy is progressing. Moreover, detectable in fetal blood, or in fetal fluids, at the time of of the endogenous hormones being produced by the the first transabdominal phase of testicular descent (Fig. 1). fetus in measurable amounts, few have the required All evidence so far indicates that the Leydig cells of the specificity to be able to offer any conclusive diagnostic testes are the only source of INSL3 in the fetus. Female outcome. So, for example, androgens are generated by fetuses consequently appear to express no or only very both adrenal glands and gonads at this time, and thus are low levels of INSL3, the origin of which is obscure expressed by both male and female fetuses in not very (Anand-Ivell, unpublished observations). Thus the detec- different amounts (Anand-Ivell et al. 2008, Scott et al. tion of INSL3 in fetal blood or amniotic fluid during 2009). An exception is provided by the small peptide mid-gestation is a very reliable indication of male fetal hormone INSL3, which is produced in large amounts gender (at least in monotocous species). Moreover, in by the fetal testis shortly after sex determination some species, e.g. cows, where maternal circulating (Anand-Ivell et al. 2008). It is produced by no other fetal organ, consequently does not appear to be Rodent F TA IS produced by the female fetus (Anand-Ivell et al. 2008, Bay et al. 2008), and is made in only very low amounts Pig F TA IS by the maternal ovary (Anand-Ivell et al. 2013). It thus Bovine F TA IS has the potential to be a highly specific monitor for any Human F TA IS events impacting on the normal differentiation and development of the fetal testis during the important window for endocrine disruption at the transition from 0 20406080100 first to second trimesters (in the human). Conception Percentage of gestation Birth Figure 1 Diagram showing relative timing of testis formation (F), as well as the transabdominal (TA) and inguino-scrotal (IS) phases of testicular INSL3 in the fetal testis descent in the human, bovine, pig, and rodent male fetus (information The fetal testis is established relatively early on during derived from various sources, including Ru¨sse & Sinowatz (1991), Hughes & Acerini (2008), and Hutson et al. (2013)). The estimated organogenesis, with the prospective somatic Sertoli and most likely ‘windows of sensitivity’ toward endocrine disruptive Leydig cells in place soon after the arrival of the agents affecting the male reproductive system are indicated by the gonocytes from the hindgut and only shortly after the red-dashed ovals.

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Downloaded from Bioscientifica.com at 09/28/2021 11:36:45PM via free access INSL3 in endocrine disruption R89 concentrations of INSL3 are low, this fetal INSL3 can in rodents, it occurs much later and is not complete until even be detected in maternal blood during mid-gestation after birth. Furthermore, fetal Leydig cell differentiation in (Anand-Ivell et al. 2011). rodents appears to occur independently of the establish- ment of the hypothalamic–pituitary–gonadal (HPG) axis (O’Shaugnessyet al.2006), unlike in humans and probably Comparative biology of testicular descent other species, which require a gonadotropin (e.g. hCG Mammals are unusual in that with a few notable or fetal luteinizing hormone (LH)) to drive Leydig cell exceptions (e.g. elephants) the male gonad does not differentiation. In the fetal pig, we had shown previously remain in a peri-renal position like the ovary, but that LHb is first evident in the pituitary on gestational day undergoes a migration, usually during fetal or early 50 but not on day 40 (Ma et al.1996), whereas INSL3 is postnatal life, to an extra-abdominal location, the already well detectable in fetal fluids on day 45 (Anand- scrotum. The reasons for this are unclear, though may Ivell, Vernunft & Ivell, unpublished observations). relate to a need for sperm in mammals to be stored at Transabdominal testicular descent, particularly in cooler than core body temperature before ejaculation those species where this occurs relatively early in ges- (Ivell 2007). This ‘testicular descent’ into the scrotum tation, such as the human, is a critical anatomical event. occurs in two phases, which may be continuous or Because it is occurring at a time of rapid fetal growth and discontinuous. The first ‘transabdominal phase’ begins in the development of other organ systems, and is direc- most species soon after sex determination (Fig. 1), and is tionally contrary to these, any disruption to the timing or achieved by INSL3 from the fetal Leydig cells acting on dynamics of gubernacular development could easily lead its specific receptor, a G--coupled receptor called to a discordant development of other organ and ligament RXFP2, which is expressed at high concentration on the systems, and vice versa (Hutson et al. 2013). It is probably cells of the gubernacular ligament (gubernacular bulb or for this reason that unilateral or inguinal cryptorchidism, gubernaculum) attached to the ventral surface of the rather than bilateral cryptorchidism, is relatively com- fetal testis and which is also linked to the abdominal mon, being caused by a relative disturbance in the growth wall of the inguinal region. INSL3 causes thickening of trajectories of different ligament systems. the gubernaculum, effectively retaining the testes in the inguinal region, while the rest of the body, including Amniotic fluid as a surrogate for measuring fetal blood the kidneys, grows away antero-dorsally. Androgens, though probably not the AMH, may Mammals, birds, and reptiles (amniotes) develop two modulate this process (Emmen et al. 2000a). The second major fluid-filled compartments to surround and protect phase of testicular descent involves the formation of the the growing fetus from which they originate. One of scrotum, the inguinal canal linking this to the peritoneal these, the allantois is best seen as an external extension cavity, and the progressive eversion of the testis together of the mesonephros and hindgut, though in humans with the gubernaculum and epididymis through the becomes rudimentary, in comparison with the amnion. canal into the scrotum (Hutson et al. 2013). This phase The amnion develops rapidly from the first through third occurs usually in the perinatal period and is regulated trimesters of pregnancy (Fig. 2) and, while early in largely by androgens (Hutson et al. 2013). gestation its fluid is contributed to mostly from the When the Insl3 gene or its receptor, RXFP2, is deleted surrounding amniotic epithelium, later in pregnancy it in mice, the major phenotype observed is a failure of becomes a reservoir for fetal urine, which, however, is testicular descent (cryptorchidism) with the testes turned over very rapidly through placental and cardio- retained in a high abdominal position (Nef & Parada vascular function (Brace 1997, Underwood et al. 2005). 1999, Zimmermann et al. 1999, Gorlov et al. 2002). This Both allantois and amnion, as well as the fetal cryptorchidism is bilateral, affecting both testes. circulation, have differing composition of amino acids Occasional defects in INSL3 production have also and electrolytes, together indicating that the fluid been found associated with cryptorchidism in dogs and contents are all under strict and independent regulation, horses (Klonisch et al. 2003, Pathirana et al. 2012). presumably as a function of their surrounding epithelial However, in humans, although cryptorchidism is rela- or endothelial layers (Goldstein et al. 1980, Brace 1997). tively common amongst newborn boys (ca. 1–7%), this Whereas, during the first and early-second trimester, is mostly transitory, unilateral, and only in rare cases amniotic fluid can also be regarded to a degree as an have any defects in the INSL3 gene or its receptor been exudate of fetal serum, later in gestation, when the fetal identified (Bogatcheva et al. 2007). skin has become keratinized, the composition of There are some important differences between species amniotic fluid changes and represents more that of in the relative timing of the first transabdominal phase of fetal urine (Underwood et al. 2005). testicular descent (Fig. 1). Whereas in humans and Importantly, amniotic fluid is accessible by needle ungulates this process and fetal testicular differentiation puncture (amniocentesis) already from the 10th week of occur relatively early in pregnancy, with the first phase gestation in humans, and thus represents one of the usually complete by the end of the second trimester; earliest points of access by which fetal health can be www.reproduction-online.org Reproduction (2014) 147 R87–R95

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1500 fetuses follows a curvilinear relationship attaining a 20 AF: blood volume ratio maximum between weeks 12 and 16, thereafter declining to below detection levels. It is not yet clear whether this decline represents a downregulation of 1000 Amniotic fluid volume INSL3 expression, or an increasing dilution effect, or (mean and quartiles) decreasing fetal skin permeability, or catabolism. The 10 only information available on INSL3 mRNA for the human fetus (O’Shaugnessy et al. 2007) suggests little 500 change in expression at least between weeks 12 and 18. Amniotic fluid volume (ml) Amniotic fluid volume AF: blood volume ratio volume blood AF: In our relatively small study, it could also be shown that amniotic INSL3 concentration was significantly Mean fetal blood volume increased for mothers who later became pre-eclamptic, 0 816243240 or reduced for those who delivered small for gestational Gestation (weeks) age babies (Anand-Ivell et al. 2008). Both of these AF INSL3 maximum symptoms are believed to have their origins in the Figure 2 Diagram showing the dynamic changes in amniotic fluid establishment of a dysfunctional placenta in the first volume and fetal blood volume through gestation in human pregnancy. trimester. Thus, these observations confirm that measure- The calculated ratio of amniotic fluid (AF):blood volumes is indicated in ment of INSL3 in amniotic fluid, because of its fetal green to estimate the possible dilution factor of INSL3 from fetal blood. specificity and its dynamic range over a short critical time The approximate time of maximum INSL3 production is shown by the window of organogenesis, can indeed act as a monitor for purple cross-hatched rectangle. Based on Smith & Cameron (2002), more general aspects of fetal health at this critical time Beall et al. (2007), and Anand-Ivell et al. (2008). point. Moreover, INSL3 is probably more effective as a monitored. Clinically, this is used to collect fetal cells parameter than, for example, steroid hormones, because suspended in amniotic fluid for prenatal genetic diag- the latter result from both fetal gonads as well as from the nosis, within a time frame which still permits legal fetal adrenal glands, and probably also the placenta, abortion if required. The supernatant from such amnio- besides possible maternal origins (Scott et al. 2009). centesis samples can also be used to assess steroid Consequently, this lack of source specificity for steroids hormones (Wudy et al. 1999, Sarkar et al. 2007) and more and hence increased variance in the system could mask recently INSL3 (Anand-Ivell et al. 2008, Bay et al. 2008). any DoHAD-related effects. Amniocentesis is routinely offered at around weeks O 12–18 of pregnancy for older women ( 35 years) at Fetal INSL3 and endocrine disruption higher risk of presenting a fetus with aneuploidy, or where there is a family genetic risk. This time point at the The linking of cryptorchidism to environmental endo- transition from the first to the second trimester coincides crine disruption, and its identification as a key symptom with the time when there should be maximal INSL3 within the TDS already predicted that, in addition to reduced androgen production, INSL3 might also be expression by the fetal testis, correlating with the first involved. This was supported by clinical studies showing transabdominal phase of testicular descent. It also that the xenoestrogen diethylstilbestrol (DES) given to represents a critical period of fetal organogenesis as pregnant mothers in the 1940s–1970s led to a high well as placental development, which are both highly incidence of cryptorchidism in the male children (Palmer susceptible to extrinsic disrupting effects (DoHAD et al. 2009), as well as the key experimental study hypothesis). We know from numerous epidemiological showing that DES given to pregnant mice caused and experimental studies that the male reproductive bilateral cryptorchidism and a downregulation of Insl3 system is indeed very vulnerable to such extrinsic effects gene expression in the fetal testis (Emmen et al. 2000b). (Sharpe & Skakkebaek 2008). Moreover, this is a time Later studies on rats then showed that the cryptorchidism period where, other than using ultrasound, no other and other TDS-like effects induced in rats by adminis- methods are routinely available to monitor fetal blood tering phthalates during a critical window of testis or fetal–maternal interaction. differentiation also led to a marked downregulation of In a preliminary study (Anand-Ivell et al. 2008), we fetal Insl3 expression (McKinnell et al. 2005, Mahood have been able to characterize INSL3 expression in et al. 2006). Combined with the knowledge from the human amniotic fluid collected at amniocentesis and mouse knockout studies, where either INSL3 or its show that maximal concentrations, which can only have receptor RXFP2 had been ablated leading to primary been derived from male fetal testes, are indeed cryptorchidism (Nef & Parada 1999, Zimmermann et al. measurable in this medium (!0.5 ng/ml). Both maternal 1999, Huang et al. 2012), this placed INSL3 firmly serum concentrations as well as amniotic fluid from within the signaling pathways, together with altered female fetuses at this time are at or below the assay limit androgen production, linking endocrine disruptors to of detection. Amniotic INSL3 concentration from male TDS-like defects – at least in rodents.

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10 gene discriminating effects of phthalates or paracetamol, whereby steroidogenesis but not INSL3 are affected. Fetal serum Amniotic fluid However, other recent studies using human adult or fetal testis explants indeed have shown that paracetamol was 8 able to impact negatively on INSL3 expression (Albert et al. 2013, Mazaud-Guittot et al. 2013). It is also important to note that such effects may also be species- 6 specific; for example, xenoestrogens may impact on INSL3 production by human fetal Leydig cells but not by those from rodents (N’Tumba-Byn et al. 2012). In a new 4 study of INSL3 expression in human amniotic fluids taken during amniocentesis, a negative correlation could be shown between INSL3 concentration and phthalate INSL3 concentration (ng/ml) INSL3 concentration load (Anand-Ivell, Toft & Jensen, unpublished obser- 2 vations), thus providing indirect evidence for an impact of endocrine disruptors on INSL3 production in the normal population. 0

GD15 GD16 GD17 GD19 GD21 INSL3 in the ‘mini-puberty’

Figure 3 INSL3 concentration in rat fetal blood serum and amniotic fluid Most mammals indicate a continual somatic deve- collected from male fetuses at various gestational days as indicated, and lopment from fetus through birth to puberty and measured using the specific rat INSL3 TRFIA as described (Anand-Ivell adulthood. Using rodents as example, we see that the et al. 2009). Unpublished data courtesy of Dr Julia Barthol (Wilmington fetal Leydig cell population involutes after birth. With DA) and Dr Ravinder Anand-Ivell (Germany). the subsequent establishment of the HPG axis, a new population of Leydig cells differentiate during puberty Firstly, in the rat, where most such environmental from resident peritubular Leydig stem cells within the disruptor studies looking at TDS symptoms have been testes (Mendis-Handagama & Ariyaratne 2001, Ge et al. carried out, Insl3 expression by the fetal Leydig cells 2006). These are the adult-type Leydig cells. In humans corresponds in time quite precisely with the so-called and higher primates, there is an unusual situation in that, ‘window of sensitivity’ that was defined for phthalate following birth, there is a prolonged period of relative exposure (Fig. 3; Carruthers & Foster 2005). Moreover, gonadal quiescence during which there is substantial immunohistochemical analysis of day 17.5 fetal rat testes exposed or not exposed to dibutyl phthalate showed maximal expression of Insl3 in Leydig cell in the controls and almost complete ablation in the phthalate-exposed 600 6 12 AF: blood volume ratio Amniotic fluid volume animals (McKinnell et al. 2005). In Fig. 3, we also show 5 10 l) parallel INSL3 measurements from both fetal blood µ and from amniotic fluid. The ratio of these two values 400 4 8 appears to correspond to the relative volumes of these two fluid compartments in this prenatal phase (Fig. 4). 3 6 But the question then arises: are the effects of phthalates

200 2 4 ratio volume blood AF: or other xenobiotics on INSL3 expression direct effects ( Amniotic fluid volume INSL3 maximum or secondary effects? Part of the difficulty here is that,

1 (g)) weight (=10% of fetal Blood volume 2 although some in vitro studies have attempted to show Fetal blood volume direct hormone action on the INSL3 gene promoter 0 0 (Lague & Tremblay 2008, 2009), other studies cannot 10 11 12 13 14 15 16 17 18 19 20 21 show this (e.g. Sadeghian et al. (2005)). Secondly, Gestation (days) concomitant with such endocrine disruptor exposures, Figure 4 Diagram showing the dynamic changes in amniotic fluid there is usually also a disruption of steroid, particularly volume and fetal blood volume through gestation in the rat. Blood androgen production. Thus effects of endocrine-disrupt- volume is estimated as 10% of fetal body weight. The calculated ing agents are more likely to be indirect via impacts on ratio of amniotic fluid (AF):blood volumes is indicated in green to Leydig cell differentiation, than directly on single estimate the possible dilution factor of INSL3 from fetal blood. like that for INSL3. Nevertheless, recent studies using The approximate time of maximum INSL3 production is shown by the purple cross-hatched rectangle. Based on Tam & Chan (1977), xenografts of human testis (Desdoits-Lethimonier et al. Park & Shepard (1994), and Anand-Ivell & Barthol (unpublished 2012) or rat fetal testis (Kristensen et al. 2012) have shown observations) (see Fig. 3). www.reproduction-online.org Reproduction (2014) 147 R87–R95

Downloaded from Bioscientifica.com at 09/28/2021 11:36:45PM via free access R92 R Anand-Ivell and R Ivell brain, skeletal, and behavioral development, with constitutively rather than being acutely regulated like puberty and the activation of the HPG axis occurring testosterone, this has been interpreted to indicate that only after several years. An exception to this is during the during puberty initially the large amounts of LH produced immediate postnatal period at ca. 3 months of age, when first cause an excessive differentiation of Leydig cells, there appears to be a short and transient upregulation of which later stabilize at a lower functional capacity androgen production by the infant testes associated with because of androgen feedback to the pituitary a brain masculinization window (Prince 2001). These (Anand-Ivell et al. 2009). A similar observation has androgens, in what is referred to as the ‘mini-puberty’, recently been found also in the dog (Pathirana et al. 2012). appear to be derived from a newly developed population Less information is available for humans and other of Leydig cells, which later regress. It is not known species. In the former, circulating INSL3 concentrations whether these neonatal Leydig cells represent a lineage have only been measured in boys up to about age of cells entirely discrete from either the fetal or adult- 15 years, where although they have attained Tanner type populations, or whether they represent a reactiva- stage 5, INSL3 concentration is evidently still increasing tion of the involuted fetal Leydig cell population. Only (Ferlin et al. 2006, Wikstro¨m et al. 2006, Johansen et al. one study has looked at INSL3 during the mini-puberty 2013). INSL3 has also been measured in adult men (Bay (Bay et al. 2007). Like testosterone, it is also elevated et al. 2005, Anand-Ivell et al. 2006), where it shows compared with later ages, showing that the Leydig cells a significant and steady decline with age from about at that time are indeed exhibiting a more mature 30 to 80 years, reflecting the gradual loss of Leydig cell phenotype. Importantly in that study a significant functionality with age (Anand-Ivell et al. 2006). What is reduction in circulating INSL3 concentration was important to note from these studies is that already at detected comparing both transiently and especially age 30 years men indicate a very broad range of INSL3 persistently cryptorchid (unilateral and bilateral) boys expression, consistent with a range of Leydig cell with normally descended controls (Bay et al. 2007). functional capacity (numbers per testis!differentiation Whether this was due to an original disruption of INSL3 status), which is likely to persist into old age and expression during fetal life, or whether this was a may determine androgen-dependent health outcomes consequence of the cryptorchid phenotype and the during aging. We have no notion as to the cause(s) maintenance of the postnatal testes at abdominal for this broad range in Leydig cell functional capacity temperature could not be determined. Interestingly, in in young men, except that it is already evident during this study expressing Leydig cell functionality as a ratio of puberty and may have its origins in some fetal or LH:INSL3 gave much more significant differences postnatal developmental disruption. between cases and controls than the ratio of In order to investigate such a possibility, studies were LH:testosterone (Bay et al. 2007). This reinforces the conducted on rats using two models of endocrine viewpoint expressed earlier that testosterone, compared disruption (Heng et al. 2012, Ivell et al. 2013). In the with INSL3, exhibits high within- and between- first model, pregnant female rats were dosed with either individual variance and consequently is less useful as a dibutyl phthalate (7!500 mg/kg body weight (BW) parameter to monitor Leydig cell function. between GD14.5 and PND6) or DES (2!125 mg/kg BW on GD14.5 and GD16.5), or appropriate vehicle, during late gestation and/or early lactation. Litters were INSL3 in the adult male then followed through puberty and male offspring Puberty in the male mammal represents the phenotypic were assessed for INSL3 secretion and expression of consequence of Leydig cell differentiation under the Leydig-cell specific genes at day 24 (during puberty) and influence of the postnatal awakening HPG axis. Pulsatile at 3 months (adulthood). Although the adult offspring GNRH leads to increased secretion of pulsatile LH from showed no obvious effects of the treatments, compared the anterior pituitary, specifically stimulating Leydig with controls, the rate of progression of puberty, in terms cells to differentiate from resident testicular stem cells of peripheral INSL3 concentration and Leydig cell and produce androgens as well as the peptide hormone numbers, and the establishment of final Leydig cell INSL3. This is well documented for INSL3 at RNA and/or functional capacity were indeed significantly disrupted secreted peptide levels in rodents (Balvers et al. 1998, (Ivell et al. 2013). In the second experiment, advantage Sadeghian et al. 2005), ruminants (Kawate et al. 2011), was taken of the property of the alkylating agent, ethane and humans (Ferlin et al. 2006, Wikstro¨m et al. 2006, dimethane sulfonate (EDS), in vivo to completely Johansen et al. 2013). Because of their relatively more eliminate all precursor and mature Leydig cells from homogeneous genetic nature, rodents exhibit a consist- the adult rat testis. Following such treatment, new Leydig ent age-dependent increase in circulating INSL3 up to a cells regenerate within the testes with a developmental maximum at the time of first appearance of sperm in the trajectory similar to that during puberty. In this study, epididymis (ca. day 42), thereafter declining somewhat similar to the previous experiment, rats were treated with to attain a lower steady state by about 6 months of age either dibutyl phthalate (500 mg/kg BW daily on days (Anand-Ivell et al. 2009). Because INSL3 is secreted 5–7 or days 3–9, following EDS injection) for a short

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Downloaded from Bioscientifica.com at 09/28/2021 11:36:45PM via free access INSL3 in endocrine disruption R93 period, or DES (125 mg/kg BW daily as for dibutyl Ethical statement phthalate), or vehicle following EDS injection, again All of the experimentation involving animals or humans, measuring INSL3 secretion and specific Leydig cell gene with which the authors have been involved and which expression during the subsequent differentiation of the is reported here, conformed to NIH guidelines, and had Leydig cells (Heng et al. 2012). As before, at day 27 after been approved by the appropriate local Animal and EDS treatment, a time equivalent to mid-puberty, there is Human Ethics Committees. a marked increase in Leydig cell numbers and in the rate of INSL3 secretion (elevated for phthalate and/or DES compared with controls), as well as in specific gene Declaration of interest expression, largely caused by a significant impact on The authors have no conflict of interest that could be perceived the rate of Leydig cell proliferation and differentiation as prejudicing the impartiality of the research reported. (Heng et al. 2012). Together, these results from rodents show that typical environmental-disrupting agents may impact not only during gestation but also on the adult Discussion from meeting phenotype during puberty or earlier. Importantly, they Luiz Franc¸a (Belo Horizonte, Brazil): In addition to its demonstrate that INSL3 measured at an appropriately role in testicular descent and apoptosis, there is dynamic time point during pubertal development can be evidence emerging that INSL3 influences spermato- very informative about effects of earlier environmental gonial stem cells and spermatogenesis. We have endocrine disruption. It should be noted, however, that preliminary evidence from zebrafish. the changes observed during Leydig cell differentiation R Ivell (Dummerstorf, Germany): There is a possible in these studies appeared to be completely resolved in role for INSL3 involvement in the testicular niche, but adulthood. This does not mean that these effects are there are as yet no solid data. without impact, as there is a disruption of endocrine Niels E Skakkebæk (Copenhagen, Denmark): Can you status at an earlier time when other organ systems are speculate about the wide range of values of INSL3 found still developing, and these might consequently be subtly in normal men in control populations. Is it better to have and chronically altered, for example, bone metabolism higher values or lower values, and what is the ideal (Ferlin et al. 2013). Although such studies use high value? Most data suggest that testicular failure and doses of potential endocrine disrupting substances, hypogonadism are associated with low INSL3, but your unlikely to be found naturally, they nevertheless point data seems compatible with higher INSL3 following fetal to aspects of developmental physiology which might exposure to endocrine disrupters. otherwise escape our attention. RIvell: More studies are required to be conclusive. Our Comparable studies in humans are obviously difficult. data show an acceleration of Leydig cell growth and However, using human adult testis explants xenografted development during rat puberty or following treatment with into immune-deficient mice, it has recently been shown ethanedimethanesulfonate (EDS). However, there are no that various analgesics in moderate doses are also able to long-term consequences in the animals followed into influence Leydig cell function in terms of INSL3 as well as adulthood. Our Australian study showed an association testosterone output (Albert et al. 2013), thus confirming between INSL3 and the metabolic syndrome, but not with also for the human that the adult testis is susceptible BMI. There is a link to bone metabolism whereby low INSL3 to endocrine disruption which can be monitored in terms is associated with osteoporosis. Smoking reduces INSL3 but of INSL3 and/or androgen production. increases testosterone. Overall it appears as if a high level of INSL3 is beneficial, but we have no longitudinal data. Bernard Je`gou (Rennes, France): We have recently Conclusions validated your hypothesis that some compounds might alter INSL3 production. In our model, we exposed adult INSL3 is a small peptide hormone which is constitutively human testes to paracetamol, aspirin, and indomethacin produced by fetal and adult Leydig cells, and by no and found inhibition of INSL3 (Albert et al., Human other tissue in appreciable amounts. Importantly, it is Reproduction, 2013). Mazz et al. (Personal commu- produced by the fetal testis during the important nicaton) showed the same effect in human fetal testes developmental window at the transition from first to exposed to paracetamol. second trimester, a time window when we know that male reproductive development is very vulnerable to a range of endocrine disrupting insults. By measuring Funding INSL3 in amniotic fluid at this time via routine Much of the research reported in this article was supported by amniocentesis, or by making use of alternative surro- the Australian Research Council, the National Health and gates, such as cord or neonatal blood, we may be able Medical Research Council of Australia, the University of to obtain significant insights into potential exposure of Adelaide, BioInnovation SA, and by the Leibniz Institute for the fetus and consequences for infant and adult health. Farm Animal Biology, Germany. This article is based on work www.reproduction-online.org Reproduction (2014) 147 R87–R95

Downloaded from Bioscientifica.com at 09/28/2021 11:36:45PM via free access R94 R Anand-Ivell and R Ivell presented at the 7th Copenhagen Workshop on Endocrine Bogatcheva NV, Ferlin A, Feng S, Truong A, Gianesello L, Foresta C & Disrupters, which was supported by the Danish Ministry of the Agoulnik AI 2007 T222P of the insulin-like 3 hormone receptor Environment – Environmental Protection Agency. Publication LGR8 is associated with testicular maldescent and hinders receptor expression on the cell surface membrane. American Journal of Physiology. of this special issue was supported by the Society for Endocrinology and Metabolism 292 E138–E144. (doi:10.1152/ajpendo. Reproduction and Fertility. Both authors received travel and 00228.2006) accommodation expenses from the Danish Ministry of the Brace RA 1997 Physiology of amniotic fluid volume regulation. Clinical Environment to attend the COW 2013 conference, the lecture Obstetrics and Gynecology 40 280–289. (doi:10.1097/00003081- at which forms the basis for this article. 199706000-00005) Carruthers CM & Foster PM 2005 Critical window of male reproductive tract development in rats following gestational exposure to di-n-butyl Acknowledgements phthalate. Birth Defects Research. Part B, Developmental and Repro- ductive Toxicology 74 277–285. (doi:10.1002/bdrb.20050) The authors gratefully acknowledge the colleagues who have De Falco T, Takahashi S & Capel B 2011 Two distinct origins for Leydig cell helped them to unravel the complexities of INSL3 expression progenitors in the fetal testis. Developmental Biology 352 14–26. (doi:10.1016/j.ydbio.2011.01.011) and its role in endocrine disruption, as well as their students for Desdoits-Lethimonier C, Albert O, Le Bizec B, Perdu E, Zalko D, Courant F, persisting with sometimes difficult projects. They thank Dr Julia Lesne L, Guille F, Dejucq-Rainsford N & Jegou B 2012 Human testis Barthol (Wilmington, DE), Drs Gunnar Toft & Morten Jensen steroidogenesis is inhibited by phthalates. Human Reproduction 27 (Aarhus, Denmark), and Dr Andreas Vernunft (Dummerstorf, 1451–1459. (doi:10.1093/humrep/des069) Germany) for permission to refer to unpublished data. Emmen JM, McLuskey A, Adham IM, Engel W, Grootegoed JA & Brinkmann AO 2000a Hormonal control of gubernaculum development during testis descent: gubernaculum outgrowth in vitro requires both insulin-like factor and androgen. Endocrinology 141 4720–4727. 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