Peter J. O’Shaughnessy

Localization of 17β-Hydroxysteroid Dehydrogenase/17-Ketosteroid Reductase Isoform Expression in the Developing Mouse Testis—Androstenedione Is the Major Androgen Secreted by Fetal/Neonatal Leydig Cells1

The final step in the biosynthesis of testosterone is reduction of androstenedione by the enzyme 17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase (17βHSD/17KSR). In this study, we have examined expression of the four known reductive isoforms of 17βHSD/17KSR (types 1, 3, 5, and 7) in the developing mouse testis and have determined changes in the localization of isoform expression and testosterone secretion during development. Using RT-PCR isoforms 1, 3, and 7 were shown to be expressed in the seminiferous tubules of neonatal testis, whereas isoforms 3 and 7 were expressed in the interstitial tissue of the adult testis. The type 7 isoform is unlikely to be involved in androgen synthesis and further study concentrated on the type 3 isoform. Developmentally, isoform type 3 was expressed in the seminiferous tubules up to day 10, showed little or no expression on day 20 and from day 30 was confined to the interstitial tissue. In situ hybridization confirmed that the type 3 isoform was expressed only in...

Effects of ACTH and expression of the melanocortin-2 receptor in the neonatal mouse testis

ACTH has been shown to stimulate androgen production by the fetal/neonatal mouse testis through the melanocortin type 2 receptor (MC2R). This study was designed to localize the expression of MC2R in the neonatal mouse testis and characterize the effects of ACTH on testicular androgen production. Using immunohistochemistry, MC2R was localized to the fetal-type Leydig cell population of the neonatal testis. ACTH caused a time-dependent increase in cyclic AMP (cAMP) and testosterone production by isolated cells with an increase in cAMP apparent in < 3 min. There was no additive effect of maximally stimulating doses of ACTH and human chorionic gonadotropin (hCG). Androgen production in response to ACTH and hCG was reduced by UO126 and dexamethasone, which are the inhibitors of ERK1/2 and phospholipase A2 respectively. Expression of mRNA encoding StAR was increased fourfold by both ACTH and hCG, although expression of mRNA encoding for steroidogenic enzymes was not markedly affected. The potency of N-terminal fragments of ACTH to stimulate androgen production was similar to that seen previously in the adrenal. Data indicate that both LH and ACTH, acting through their respective receptors, stimulate steroidogenesis by fetal-type Leydig cells via arachidonic acid, protein kinase A, and ERK1/2 activation of StAR.

The human placental proteome is affected by maternal smoking

Highlights • The effects of maternal smoking on the term placental proteome was studied.• Maternal smoking significantly affected 18% of protein spots.• Maternal smoking affects systems controlling the development and function of placenta.• The observed placental changes may contribute to the lowered birth weights.

Chapter 14 – Testicular Development

This chapter describes our current understanding of testis development and the mechanisms involved, and highlights some of the clear gaps in our knowledge. The period of testis development between initial differentiation and puberty is critical for ensuring masculinization of the fetus, adult fertility, and, potentially, for adult health and well-being. During this period, Sertoli cell numbers (and, thus, germ cell numbers) are established, two populations of Leydig cells arise, and spermatogonial differentiation occurs. In most species the testes develop in the first third of gestation and go through a period of hormone-independent development. By about the last third of gestation, however, androgen secretion by the fetal Leydig cells and Sertoli cell proliferation are both dependent upon the pituitary gonadotropins. After birth the fetal Leydig cells are superseded by a second, adult population of cells that secretes androgen under the stimulation of luteinizing hormone. Continued Sertoli cell proliferation through late fetal development and in the neonate is largely dependent upon androgen secreted by the Leydig cells and upon follicle-stimulating hormone. The Sertoli cells stop proliferating just prior to puberty and, at that stage, establish the phenotypic activity of the adult cells. This, in turn, allows germ cell development to progress through meiosis.

Development of Leydig Cell Steroidogenesis

During development two or three distinct populations of Leydig cells arise sequentially. These cell populations appear to represent distinct cell lineages and show clear differences in their regulation and pattern of development. The fetal Leydig cells arise soon after testis differentiation and appear to go through an early phase of hormone-independent development where paracrine factors or constitutive activity may regulate cell function. Thereafter, in the mouse, the cells become dependent on pituitary hormone action and current evidence suggests that both luteinizing hormone (LH) and adrenocorticotrophic hormone may regulate development. The adult population of Leydig cells arises before puberty, around day 7 in the mouse. Factors initiating and regulating this development are unclear but LH does not appear to play an early role in adult Leydig cell differentiation. Following differentiation, there is a marked proliferation of Leydig cell number before puberty although the cells do not become steroidogenically active until near puberty. Both proliferation and functional development of the adult Leydig cell population are critically dependent on LH although, in the mouse, androgens are also essential for normal Leydig cell development.

Testicular cell selective ablation using diphtheria toxin receptor transgenic mice

Testis development and function is regulated by intricate cell-cell cross talk. Characterization of the mechanisms underpinning this has been derived through a wide variety of approaches including pharmacological manipulation, transgenics, and cell-specific ablation of populations. The removal of all or a proportion of a specific cell type has been achieved through a variety of approaches. In this paper, we detail a combined transgenic and pharmacological approach to ablate the Sertoli or germ cell populations using diphtheria toxin in mice. We describe the key steps in generation, validation, and use of the models and also describe the caveats and cautions necessary. We also provide a detailed description of the methodology applied to characterize testis development and function in models of postnatal Sertoli or germ cell ablation.

Modelling steroidogenesis: a framework model to support hypothesis generation and testing across endocrine studies

ObjectiveSteroid hormones are responsible for the control of a wide range of physiological processes such as development, growth, reproduction, metabolism, and aging. Because of the variety of enzymes, substrates and products that take part in steroidogenesis and the compartmentalisation of its constituent reactions, it is a complex process to visualise and document. One of the goals of systems biology is to quantitatively describe the behaviour of complex biological systems that involve the interaction of many components. This can be done by representing these interactions visually in a pathway model and then optionally constructing a mathematical model of the interactions.ResultsWe have used the modified Edinburgh Pathway Notation to construct a framework diagram describing human steroidogenic pathways, which will be of use to endocrinologists. To demonstrate further utility, we show how such models can be parameterised with empirical data within the software Graphia Professional, to recapitulate specific examples of steroid hormone production, and also to mimic gene knockout. These framework models support in silico hypothesis generation and testing with utility across endocrine endpoints, with significant potential to reduce costs, time and animal numbers, whilst informing the design of planned studies.

Maternal smoking and high BMI disrupt thyroid gland development

BackgroundMaternal lifestyle factors, including smoking and increased body weight, increase risks of adult diseases such as metabolic syndrome and infertility. The fetal thyroid gland is essential for the control of fetal metabolic rate, cardiac output, and brain development. Altered fetal thyroid function may contribute to increased disease onset later in life. Here, we investigated the impact of maternal smoking and high maternal weight on human fetal thyroid function during the second trimester.MethodsThyroid glands and plasma were collected from fetuses electively terminated in the second trimester (normally progressing pregnancies). Plasma total triiodothyronine (T3) and total thyroxine (T4) were measured by solid-phase extraction-liquid chromatography-tandem mass spectrometry. Fetal plasma thyroid-stimulating hormone (TSH) levels were measured using a multiplex assay for human pituitary hormones. Histology and immunolocalization of thyroid developmental markers were examined in thyroid sections. Transcript levels of developmental, functional, apoptotic, and detoxification markers were measured by real-time PCR. Statistical analyses were performed using multivariate linear regression models with fetal age, sex, and maternal smoking or maternal body mass index (BMI) as covariates.ResultsMaternal smoking was associated with significant changes in fetal plasma T4 and TSH levels during the second trimester. Smoke-exposed thyroids had reduced thyroid GATA6 and NKX2-1 transcript levels and altered developmental trajectories for ESR2 and AHR transcript levels. Maternal BMI > 25 was associated with increased fetal thyroid weight, increased plasma TSH levels, and abnormal thyroid histology in female fetuses. Normal developmental changes in AHR and ESR1 transcript expression were also abolished in fetal thyroids from mothers with BMI > 25.ConclusionsFor the first time, we show that maternal smoking and high maternal BMI are associated with disturbed fetal thyroid gland development and endocrine function in a sex-specific manner during the second trimester. These findings suggest that predisposition to post-natal disease is mediated, in part, by altered fetal thyroid gland development.

The Human Leydig Cell

The Leydig cells are found in the interstitial compartment of the testis and are the major source of androgens in males. At least two populations of Leydig cells differentiate sequentially as the testis develops—a fetal population which regulates masculinization in utero and an adult population which develops before puberty and regulates adult fertility and sex drive. A third, neonatal population is also observed in the human which may represent re-activation of the fetal Leydig cells. The fetal Leydig cells in the human depend upon stimulation by chorionic gonadotropin and produce androgens through the canonical steroidogenic pathway and also, possibly, through an alternative “backdoor” pathway; both pathways apparently being required for normal fetal masculinization. Fetal Leydig cells also secrete insulin-like factor 3 (INSL3) which, along with androgen, induces testicular descent. The fetal Leydig cell population persists into adulthood in mice but becomes secondary to the adult population. Development of the adult Leydig cell population is dependent on the Sertoli cells and on luteinizing hormone (LH) from the pituitary. The adult cells in humans secrete mainly testosterone synthesized through the Δ5 canonical pathway, and cell activity is dependent on LH and the bone-derived hormone osteocalcin while the Sertoli cells, through unknown factors, act to maintain Leydig cell numbers. During aging in humans, there is a reduction in Leydig cell activity and, possibly, Leydig cell numbers. Leydig cell tumors are rare but will lead to precocious puberty when they occur in prepubertal boys. In about half of cases these tumors are associated with activating mutations in the steroidogenic machinery [e.g., the luteinizing hormone/choriogonadotropin receptor (LHCGR)].

Erratum to: Modelling foetal exposure to maternal smoking using hepatoblasts from pluripotent stem cells

During manuscript proofing, the following sentence was not deleted in the section “Results” at the end of the paragraph: “Both male and female hepatocytes responded in a similar fashion to cotinine, whereas male hepatocyte function was more sensitive to chrysene, fluorene and naphthalene than female hepatocytes”.