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 cryptorchidism. 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 gene 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 hormones 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 hormone-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 & peptide 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).