Richard M. Sharpe

Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract

The incidence of disorders of development of the male reproductive tract has more than doubled in the past 30-50 years while sperm counts have declined by about half. Similar abnormalities occur in the sons of women exposed to diethylstilbestrol (DES) during pregnancy and can be induced in animals by brief exposure to exogenous oestrogen/DES during pregnancy. We argue that the increasing incidence of reproductive abnormalities in the human male may be related to increased oestrogen exposure in utero, and identify mechanisms by which this exposure could occur.

Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism

Becoming a phenotypic male is ultimately determined by androgen-induced masculinization. Disorders of fetal masculinization, resulting in hypospadias or cryptorchidism, are common, but their cause remains unclear. Together with the adult-onset disorders low sperm count and testicular cancer, they can constitute a testicular dysgenesis syndrome (TDS). Although masculinization is well studied, no unifying concept explains normal male reproductive development and its abnormalities, including TDS. We exposed rat fetuses to either anti-androgens or androgens and showed that masculinization of all reproductive tract tissues was programmed by androgen action during a common fetal programming window. This preceded morphological differentiation, when androgen action was, surprisingly, unnecessary. Only within the programming window did blocking androgen action induce hypospadias and cryptorchidism and altered penile length in male rats, all of which correlated with anogenital distance (AGD). Androgen-driven masculinization of females was also confined to the same programming window. This work has identified in rats a common programming window in which androgen action is essential for normal reproductive tract masculinization and has highlighted that measuring AGD in neonatal humans could provide a noninvasive method to predict neonatal and adult reproductive disorders. Based on the timings in rats, we believe the programming window in humans is likely to be 8-14 weeks of gestation.

Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects

Reproductive disorders of newborn (cryptorchidism, hypospadias) and young adult males (low sperm counts, testicular germ cell cancer) are common and/or increasing in incidence. It has been hypothesized that these disorders may comprise a testicular dysgenesis syndrome (TDS) with a common origin in fetal life. This has been supported by findings in an animal model of TDS involving fetal exposure to n(dibutyl) phthalate, as well as by new clinical studies. Recent advances in understanding from such studies have led to refinement of the TDS hypothesis, highlighting the central role that deficient androgen production/action during fetal testis development, may play in the origin of downstream disorders.

Public Health Implications of Altered Puberty Timing

Changes in puberty timing have implications for the treatment of individual children, for the risk of later adult disease, and for chemical testing and risk assessment for the population. Children with early puberty are at a risk for accelerated skeletal maturation and short adult height, early sexual debut, potential sexual abuse, and psychosocial difficulties. Altered puberty timing is also of concern for the development of reproductive tract cancers later in life. For example, an early age of menarche is a risk factor for breast cancer. A low age at male puberty is associated with an increased risk for testicular cancer according to several, but not all, epidemiologic studies. Girls and, possibly, boys who exhibit premature adrenarche are at a higher risk for developing features of metabolic syndrome, including obesity, type 2 diabetes, and cardiovascular disease later in adulthood. Altered timing of puberty also has implications for behavioral disorders. For example, an early maturation is associated with a greater incidence of conduct and behavior disorders during adolescence. Finally, altered puberty timing is considered an adverse effect in reproductive toxicity risk assessment for chemicals. Recent US legislation has mandated improved chemical testing approaches for protecting childrens health and screening for endocrine-disrupting agents, which has led to changes in the US Environmental Protection Agencys risk assessment and toxicity testing guidelines to include puberty-related assessments and to the validation of pubertal male and female rat assays for endocrine screening.

Permanent Effects of Neonatal Estrogen Exposure in Rats on Reproductive Hormone Levels, Sertoli Cell Number, and the Efficiency of Spermatogenesis in Adulthood*

This study aimed to identify the mechanism(s) for impairment of spermatogenesis in adulthood in rats treated neonatally with estrogens. Rats were treated (days 2-12) with 10, 1, or 0.1 microg diethylstilbestrol (DES), 10 microg ethinyl estradiol (EE), 10 mg/kg of a GnRH antagonist (GnRHa), or vehicle and killed in adulthood. DES/EE caused dose-dependent reductions in testis weight, total germ cell volume per testis, and Sertoli cell volume per testis. Sertoli cell number at 18 days of age in DES-treated rats was reduced dose dependently. GnRHa treatment caused changes in these parameters similar to those in rats treated with 10 microg DES. Plasma FSH levels were elevated (P < 0.001) to similar levels in all treatment groups regardless of differences in Sertoli cell number and levels of inhibin B; the latter reflected Sertoli cell number, but levels were disproportionately reduced in animals treated with high doses of DES/EE. Neonatal estrogen treatment, but not GnRHa, caused dose-dependent reductions (40-80%) in plasma testosterone levels in adulthood, but did not alter LH levels. Preliminary evidence suggests that the decrease in testosterone levels in estrogen-treated rats is not due to reduced Leydig cell volume per testis. GnRHa-treated rats exhibited a significant increase in germ cell volume per Sertoli cell and a reduction in germ cell apoptosis, probably because of the raised FSH levels. Despite similar raised FSH levels, rats treated with DES (10 or 1 microg) or EE (10 microg) had reduced germ cell volume/Sertoli cell and increased germ cell apoptosis, especially when compared with GnRHa-treated animals. The latter changes were associated with an increase in lumen size per testis, indicative of impaired fluid resorption from the efferent ducts, resulting in fluid accumulation in the testis. Rats treated neonatally with 0.1 microg DES showed reduced germ cell apoptosis comparable to that in GnRHa-treated animals. The changes in apoptotic rate among treatment groups occurred across all stages of the spermatogenic cycle. It is concluded that 1) neonatal estrogen treatment results in dose-dependent alterations in Sertoli cell numbers, germ cell volume, efficiency of spermatogenesis, and germ cell apoptosis in adulthood; 2) the relatively poor spermatogenesis in estrogen-treated animals is most likely due to altered testis fluid dynamics and/or altered Sertoli cell function; 3) as indicated by FSH (LH) and testosterone levels, the hypothalamic-pituitary axis and Leydig cells are probably more sensitive than the Sertoli cells to reprogramming by estrogens neonatally; and 4) elevated FSH levels in adulthood may improve the efficiency of spermatogenesis.

Environmental/lifestyle effects on spermatogenesis

The high incidence of low sperm counts in young (European) men and evidence for declining sperm counts in recent decades mean that the environmental/lifestyle impact on spermatogenesis is an important health issue. This review assesses potential causes involving adverse effects on testis development in perinatal life (primarily effects on Sertoli cell number), which are probably irreversible, or effects on the process of spermatogenesis in adulthood, which are probably mainly reversible. Several lifestyle-related (obesity, smoking) and environmental (exposure to traffic exhaust fumes, dioxins, combustion products) factors appear to negatively affect both the perinatal and adult testes, emphasizing the importance of environmental/lifestyle impacts throughout the life course. Apart from this, public concern about adverse effects of environmental chemicals (ECs) (pesticides, food additives, persistent pollutants such as DDT, polychlorinated biphenyls) on spermatogenesis in adult men are, in general, not supported by the available data for humans. Where adverse effects of ECs have been shown, they are usually in an occupational setting rather than applying to the general population. In contrast, a modern Western lifestyle (sedentary work/lifestyle, obesity) is potentially damaging to sperm production. Spermatogenesis in normal men is poorly organized and inefficient so that men are poorly placed to cope with environmental/lifestyle insults.

Rodent Leydig cell tumorigenesis: a review of the physiology, pathology, mechanisms, and relevance to humans.

Leydig cells (LCs) are the cells of the testis that have as their primary function the production of testosterone. LCs are a common target of compounds tested in rodent carcinogenicity bioassays. The number of reviews on Leydig cell tumors (LCTs) has increased in recent years because of its common occurrence in rodent bioassays and the importance in assessing the relevance of this tumor type to humans. To date, there have been no comprehensive reviews to identify all the compounds that have been shown to induce LCTs in rodents or has any review systematically evaluated the epidemiology data to determine whether humans were at increased risk for developing LCTs from exposure to these agents. This review attempts to fill these deficiencies in the literature by comparing the cytology and ontogeny of the LC, as well as the endocrine and paracrine regulation of both normal and tumorigenic LCs. In addition, the pathology of LCTs in rodents and humans is compared, compounds that induce LC hyperplasia or tumors are enumerated, and the human relevance of chemical-induced LCTs is discussed. There are plausible mechanisms for the chemical induction of LCTs, as typified by agonists of estrogen, gonadotropin releasing hormone (GnRH), and dopamine receptors, androgen receptor antagonists, and inhibitors of 5alpha-reductase, testosterone biosynthesis, and aromatase. Most of these ultimately involve elevation in serum luteinizing hormone (LH) and/or LC responsiveness to LH as proximate mediators. It is expected that further work will uncover additional mechanisms by which LCTs may arise, especially the role of growth factors in modulating LC tumorigenesis. Regarding human relevance, the pathways for regulation of the hypothalamo-pituitary-testis (HPT) axis of rats and humans are similar, such that compounds that either decrease testosterone or estradiol levels or their recognition will increase LH levels. Hence, compounds that induce LCTs in rats by disruption of the HPT axis pose a risk to human health, except for possibly two classes of compounds (GnRH and dopamine agonists). Because GnRH and prolactin receptors are either not expressed or are expressed at very low levels in the testes in humans, the induction of LCTs in rats by GnRH and dopamine agonists would appear not to be relevant to humans; however, the potential relevance to humans of the remaining five pathways of LCT induction cannot be ruled out. Therefore, the central issue becomes what is the relative sensitivity between rat and human LCs in their response to increased LH levels; specifically, is the proliferative stimulus initiated by increased levels of LH attenuated, similar, or enhanced in human vs. rat LCs? There are several lines of evidence that suggest that human LCs are quantitatively less sensitive than rats in their proliferative response to LH, and hence in their sensitivity to chemically induced LCTs. This evidence includes the following: (1) the human incidence of LCTs is much lower than in rodents even when corrected for detection bias; (2) several comparative differences exist between rat and human LCs that may contribute, at least in part, to the greater susceptibility of the rat to both spontaneous and xenobiotic-induced LCTs; (3) endocrine disease states in man (such as androgen-insensitivity syndrome and familial male precocious puberty) underscore the marked comparative differences that exist between rats and man in the responsiveness of their LCs to proliferative stimuli; and (4) several human epidemiology studies are available on a number of compounds that induce LCTs in rats (1,3-butadiene, cadmium, ethanol, lactose, lead, nicotine) that demonstrate no association between human exposure to these compounds and induction of LC hyperplasia or adenomas. (ABSTRACT TRUNCATED)

Dietary soy isoflavone-induced increases in antioxidant and eNOS gene expression lead to improved endothelial function and reduced blood pressure in vivo

Epidemiological evidence suggests that populations consuming large amounts of soy protein have a reduced incidence of coronary heart disease (1–5). The cardiovascular risks associated with conventional hormone replacement therapy in postmenopausal women (5–7) have precipitated a search for alternative estrogen receptor modulators. Here we report that long‐term feeding of rats with a soy protein‐rich (SP) diet during gestation and adult life results in decreased oxidative stress, improved endothelial function, and reduced blood pressure in vivo measured by radiotelemetry in aged male offspring. Improved vascular reactivity in animals fed an SP diet was paralleled by increased mitochondrial glutathione and mRNA levels for endothelial nitric oxide synthase (eNOS) and the antioxidant enzymes manganese superoxide dismutase and cytochrome c oxidase. Reduced eNOS and antioxidant gene expression, impaired endothelial function, and elevated blood pressure in animals fed a soy‐deficient diet was reversed after refeeding them an SP diet for 6 months. Our findings suggest that an SP diet increases eNOS and antioxidant gene expression in the vasculature and other tissues, resulting in reduced oxidative stress and increased NO bioavailability. The improvement in endothelial function, increased gene expression, and reduced blood pressure by soy isoflavones have implications for alternative therapy for postmenopausal women and patients at risk of coronary heart disease.

Androgen action via testicular peritubular myoid cells is essential for male fertility

Androgens are essential for normal spermatogenesis and male fertility, but how androgens exert this effect remains uncertain. Androgen receptors (ARs) are expressed in several testicular cell types, but continuing uncertainty exists over which cell type mediates androgen control of spermatogenesis. Androgen signaling via Sertoli cells (SCs) is essential for complete spermatogen‐esis, but the role for androgen signaling via peritubular myoid (PTM) cells is contentious. To address this controversy, we generated PTM‐specific AR‐knockout (PTM‐ARKO) mice in which gross reproductive development was normal, but all PTM‐ARKO males were azoospermic and infertile. Testis weight was reduced beyond puberty, and in adulthood there was an 86% reduction in germ cells, compared with wild‐type littermates. These changes were not explained by any deficits in testosterone, lutein‐izing hormone, or follicle‐stimulating hormone concentrations. SC function was impaired in PTM‐ARKO males, indicated by reduced seminiferous tubule fluid production and reduced expression of some androgen‐depen‐dent SC genes. Androgen action via PTM cells is therefore essential for normal testis function, spermatogenesis, and fertility in males. This study also provides the first direct evidence for the importance of androgen‐driven stromal‐epithelial interactions underpinning the regulation of spermatogenesis; PTM‐ARKO mice will enable identification of the new molecular pathways involved.— Welsh, M., Saunders, P. T. K., Atanassova, N., Sharpe, R. M., Smith, L. B. Androgen action via testicular peritu‐bular myoid cells is essential for male fertility. FASEB J. 23, 4218‐4230 (2009). www.fasebj.org

Effects of monobutyl and di(n-butyl) phthalate in vitro on steroidogenesis and Leydig cell aggregation in fetal testis explants from the rat: comparison with effects in vivo in the fetal rat and neonatal marmoset and in vitro in the human.

Background Certain phthalates can impair Leydig cell distribution and steroidogenesis in the fetal rat in utero, but it is unknown whether similar effects might occur in the human. Objectives Our aim in this study was to investigate the effects of di(n-butyl) phthalate (DBP), or its metabolite monobutyl phthalate (MBP), on testosterone production and Leydig cell aggregation (LCA) in fetal testis explants from the rat and human, and to compare the results with in vivo findings for DBP-exposed rats. We also wanted to determine if DBP/MBP affects testosterone production in vivo in the neonatal male marmoset. Methods Fetal testis explants obtained from the rat [gestation day (GD)19.5] and from the human (15–19 weeks of gestation) were cultured for 24–48 hr with or without human chorionic gonadotropin (hCG) or 22R-hydroxycholesterol (22R-OH), and with or without DBP/MBP. Pregnant rats and neonatal male marmosets were dosed with 500 mg/kg/day DBP or MBP. Results Exposure of rats in utero to DBP (500 mg/kg/day) for 48 hr before GD21.5 induced major suppression of intratesticular testosterone levels and cytochrome P450 side chain cleavage enzyme (P450scc) expression; this short-term treatment induced LCA, but was less marked than longer term (GD13.5–20.5) DBP treatment. In vitro, MBP (10−3 M) did not affect basal or 22R-OH-stimulated testosterone production by fetal rat testis explants but slightly attenuated hCG-stimulated steroidogenesis; MBP induced minor LCA in vitro. None of these parameters were affected in human fetal testis explants cultured with 10−3 M MBP for up to 48 hr. Because the in vivo effects of DBP/MBP were not reproduced in vitro in the rat, the absence of MBP effects in vitro on fetal human testes is inconclusive. In newborn (Day 2–7) marmosets, administration of a single dose of 500 mg/kg MBP significantly (p = 0.019) suppressed blood testosterone levels 5 hr later. Similar treatment of newborn co-twin male marmosets for 14 days resulted in increased Leydig cell volume per testis (p = 0.011), compared with co-twin controls; this is consistent with MBP-induced inhibition of steroidogenesis followed by compensatory Leydig cell hyperplasia/hypertrophy. Conclusions These findings suggest that MBP/DBP suppresses steroidogenesis by fetal-type Leydig cells in primates as in rodents, but this cannot be studied in vitro.