Endocrine disruption and male reproductive disorders: unanswered questions.

Maternal exposure to endocrine-disrupting chemicals (EDCs) in human pregnancy is widely considered as an important cause of adverse changes in male reproductive health due to impaired foetal androgen production/action. However, the epidemiological evidence supporting this view is equivocal, except for certain phthalates, notably diethyl hexyl phthalate (DEHP). Maternal phthalate exposure levels associated with adverse reproductive changes in epidemiological studies are several thousand-fold lower than those needed to suppress foetal androgen production in rats, and direct studies using human foetal testis tissue show no effect of high phthalate exposure on androgen production. This conundrum is unexplained and raises fundamental questions. Human DEHP exposure is predominantly via food with highest exposure associated with consumption of a Western style (unhealthy) diet. This diet is also associated with increased exposure to the most common EDCs, whether persistent (chlorinated or fluorinated chemicals) or non-persistent (phthalates, bisphenols) compounds, which are found at highest levels in fatty and processed foods. Consequently, epidemiological studies associating EDC exposure and male reproductive health disorders are confounded by potential dietary effects, and vice versa. A Western diet/lifestyle in young adulthood is also associated with low sperm counts. Disentangling EDC and dietary effects in epidemiological studies is challenging. In pregnancy, a Western diet, EDC exposure, and maternal living in proximity to industrial sites are all associated with impaired foetal growth/development due to placental dysfunction, which predisposes to congenital male reproductive disorders (cryptorchidism, hypospadias). While the latter are considered to reflect impaired foetal androgen production, effects resulting from foetal growth impairment (FGI) are likely indirect. As FGI has numerous life-long health consequences, and is affected by maternal lifestyle, research into the origins of male reproductive disorders should take more account of this. Additionally, potential effects on foetal growth/foetal testis from the increasing use of medications in pregnancy deserves more research attention.

Draft genome data of Prunus avium cv 'Stella'.

Prunus avium cv. 'Stella' total cellular DNA was isolated from emerging leaf tissue and sequenced using Roche 454 GS FLX Titanium, and Illumina HiSeq 2000 High Throughput Sequencing (HTS) technologies. Sequence data were filtered and trimmed to retain nucleotides corresponding to Phred score 30, and assembled with CLC Genomics Workbench v.6.0.1. A total of 107,531 contigs were assembled with 185 scaffolds with a maximum length of 132,753 nucleotides and an N50 value of 4,601. The average depth of coverage was 135.87 nucleotides with a median depth of coverage equal to 31.50 nucleotides. The draft 'Stella' genome presented here covers 77.8% of the estimated 352.9Mb P. avium genome and is expected to facilitate genetics and genomics research focused on identifying genes and quantitative trait loci (QTL) underlying important agronomic and consumer traits.

Identification of Root Rot Resistance QTLs in Pea Using Fusarium solani f. sp. pisi-Responsive Differentially Expressed Genes.

Pisum sativum (pea) yields in the United States have declined significantly over the last decades, predominantly due to susceptibility to root rot diseases. One of the main causal agents of root rot is the fungus Fusarium solani f. sp. pisi (Fsp), leading to yield losses ranging from 15 to 60%. Determining and subsequently incorporating the genetic basis for resistance in new cultivars offers one of the best solutions to control this pathogen; however, no green-seeded pea cultivars with complete resistance to Fsp have been identified. To date, only partial levels of resistance to Fsp has been identified among pea genotypes. SNPs mined from Fsp-responsive differentially expressed genes (DEGs) identified in a preceding study were utilized to identify QTLs associated with Fsp resistance using composite interval mapping in two recombinant inbred line (RIL) populations segregating for partial root rot resistance. A total of 769 DEGs with single nucleotide polymorphisms (SNPs) were identified, and the putative SNPs were evaluated for being polymorphic across four partially resistant and four susceptible P. sativum genotypes. The SNPs with validated polymorphisms were used to screen two RIL populations using two phenotypic criteria: root disease severity and plant height. One QTL, WB.Fsp-Ps 5.1 that mapped to chromosome 5 explained 14.8% of the variance with a confidence interval of 10.4 cM. The other four QTLs located on chromosomes 2, 3, and 5, explained 5.3-8.1% of the variance. The use of SNPs derived from Fsp-responsive DEGs for QTL mapping proved to be an efficient way to identify molecular markers associated with Fsp resistance in pea. These QTLs are potential candidates for marker-assisted selection and gene pyramiding to obtain high levels of partial resistance in pea cultivars to combat root rot caused by Fsp.

Estrogens and development of the rete testis, efferent ductules, epididymis and vas deferens.

Estrogen has always been considered the female hormone and testosterone the male hormone. However, estrogen's presence in the testis and deleterious effects of estrogen treatment during development have been known for nearly 90 years, long before estrogen receptors (ESRs) were discovered. Eventually it was learned that testes actually synthesize high levels of estradiol (E2) and sequester high concentrations in the reproductive tract lumen, which seems contradictory to the overwhelming number of studies showing reproductive pathology following exogenous estrogen exposures. For too long, the developmental pathology of estrogen has dominated our thinking, even resulting in the "estrogen hypothesis" as related to the testicular dysgenesis syndrome. However, these early studies and the development of an Esr1 knockout mouse led to a deluge of research into estrogen's potential role in and disruption of development and function of the male reproductive system. What is new is that estrogen action in the male cannot be divorced from that of androgen. This paper presents what is known about components of the estrogen pathway, including its synthesis and target receptors, and the need to achieve a balance between androgen- and estrogen-action in male reproductive tract differentiation and adult functions. The review focuses on what is known regarding development of the male reproductive tract, from the rete testis to the vas deferens, and examines the expression of estrogen receptors and presence of aromatase in the male reproductive system, traces the evidence provided by estrogen-associated knockout and transgenic animal models and discusses the effects of fetal and postnatal exposures to estrogens. Hopefully, there will be enough here to stimulate discussions and new investigations of the androgen:estrogen balance that seems to be essential for development of the male reproductive tract.

Identification of Fusarium solani f. sp. pisi (Fsp) Responsive Genes in Pisum sativum.

Pisum sativum (pea) is rapidly emerging as an inexpensive and significant contributor to the plant-derived protein market. Due to its nitrogen-fixation capability, short life cycle, and low water usage, pea is a useful cover-and-break crop that requires minimal external inputs. It is critical for sustainable agriculture and indispensable for future food security. Root rot in pea, caused by the fungal pathogen Fusarium solani f. sp. pisi (Fsp), can result in a 15-60% reduction in yield. It is urgent to understand the molecular basis of Fsp interaction in pea to develop root rot tolerant cultivars. A complementary genetics and gene expression approach was undertaken in this study to identify Fsp-responsive genes in four tolerant and four susceptible pea genotypes. Time course RNAseq was performed on both sets of genotypes after the Fsp challenge. Analysis of the transcriptome data resulted in the identification of 42,905 differentially expressed contigs (DECs). Interestingly, the vast majority of DECs were overexpressed in the susceptible genotypes at all sampling time points, rather than in the tolerant genotypes. Gene expression and GO enrichment analyses revealed genes coding for receptor-mediated endocytosis, sugar transporters, salicylic acid synthesis, and signaling, and cell death were overexpressed in the susceptible genotypes. In the tolerant genotypes, genes involved in exocytosis, and secretion by cell, the anthocyanin synthesis pathway, as well as the DRR230 gene, a pathogenesis-related (PR) gene, were overexpressed. The complementary genetic and RNAseq approach has yielded a set of potential genes that could be targeted for improved tolerance against root rot in P. sativum. Fsp challenge produced a futile transcriptomic response in the susceptible genotypes. This type of response is hypothesized to be related to the speed at which the pathogen infestation advances in the susceptible genotypes and the preexisting level of disease-preparedness in the tolerant genotypes.

Methods of analysis of chloroplast genomes of C3, Kranz type C4 and Single Cell C4 photosynthetic members of Chenopodiaceae.

BACKGROUND: Chloroplast genome information is critical to understanding forms of photosynthesis in the plant kingdom. During the evolutionary process, plants have developed different photosynthetic strategies that are accompanied by complementary biochemical and anatomical features. Members of family Chenopodiaceae have species with C3 photosynthesis, and variations of C4 photosynthesis in which photorespiration is reduced by concentrating CO2 around Rubisco through dual coordinated functioning of dimorphic chloroplasts. Among dicots, the family has the largest number of C4 species, and greatest structural and biochemical diversity in forms of C4 including the canonical dual-cell Kranz anatomy, and the recently identified single cell C4 with the presence of dimorphic chloroplasts separated by a vacuole. This is the first comparative analysis of chloroplast genomes in species representative of photosynthetic types in the family. RESULTS: Methodology with high throughput sequencing complemented with Sanger sequencing of selected loci provided high quality and complete chloroplast genomes of seven species in the family and one species in the closely related Amaranthaceae family, representing C3, Kranz type C4 and single cell C4 (SSC4) photosynthesis six of the eight chloroplast genomes are new, while two are improved versions of previously published genomes. The depth of coverage obtained using high-throughput sequencing complemented with targeted resequencing of certain loci enabled superior resolution of the border junctions, directionality and repeat region sequences. Comparison of the chloroplast genomes with previously sequenced plastid genomes revealed similar genome organization, gene order and content with a few revisions. High-quality complete chloroplast genome sequences resulted in correcting the orientation the LSC region of the published Bienertia sinuspersici chloroplast genome, identification of stop codons in the rpl23 gene in B. sinuspersici and B. cycloptera, and identifying an instance of IR expansion in the Haloxylon ammodendron inverted repeat sequence. The rare observation of a mitochondria-to-chloroplast inter-organellar gene transfer event was identified in family Chenopodiaceae. CONCLUSIONS: This study reports complete chloroplast genomes from seven Chenopodiaceae and one Amaranthaceae species. The depth of coverage obtained using high-throughput sequencing complemented with targeted resequencing of certain loci enabled superior resolution of the border junctions, directionality, and repeat region sequences. Therefore, the use of high throughput and Sanger sequencing, in a hybrid method, reaffirms to be rapid, efficient, and reliable for chloroplast genome sequencing.

Androgens and the masculinization programming window: human-rodent differences.

Human male reproductive disorders are common and may have a fetal origin - the testicular dysgenesis syndrome (TDS) hypothesis. In rats, experimentally induced TDS disorders result from disruption of fetal androgen production/action specifically in the masculinization programming window (MPW). MPW androgen action also programs longer anogenital distance (AGD) in male versus female rats; shorter male AGD is correlated with risk and severity of induced TDS disorders. AGD thus provides a lifelong, calibrated readout of MPW androgen exposure and predicts likelihood of reproductive dysfunction. Pregnant rat exposure to environmental chemicals, notably certain phthalates (e.g. diethyl hexl phthalate, DEHP; dibutyl phthalate, DBP), pesticides or paracetamol, can reduce fetal testis testosterone and AGD and induce TDS disorders, provided exposure includes the MPW. In humans, AGD is longer in males than females and the presumptive MPW is 8-14 weeks' gestation. Some, but not all, epidemiological studies of maternal DEHP (or pesticides) exposure reported shorter AGD in sons, but this occurred at DEHP exposure levels several thousand-fold lower than are effective in rats. In fetal human testis culture/xenografts, DEHP/DBP do not reduce testosterone production, whereas therapeutic paracetamol exposure does. In humans, androgen production in the MPW is controlled differently (human chorionic gonadotrophin-driven) than in rats (paracrine controlled), and other organs (placenta, liver, adrenals) contribute to MPW androgens, essential for normal masculinization, via the 'backdoor pathway'. Consequently, early placental dysfunction, which is affected by maternal lifestyle and diet, and maternal painkiller use, may be more important than environmental chemical exposures in the origin of TDS in humans.

Concomitant phytonutrient and transcriptome analysis of mature fruit and leaf tissues of tomato (Solanum lycopersicum L. cv. Oregon Spring) grown using organic and conventional fertilizer.

Enhanced levels of antioxidants, phenolic compounds, carotenoids and vitamin C have been reported for several crops grown under organic fertilizer, albeit with yield penalties. As organic agricultural practices continue to grow and find favor it is critical to gain an understanding of the molecular underpinnings of the factors that limit the yields in organically farmed crops. Concomitant phytochemical and transcriptomic analysis was performed on mature fruit and leaf tissues derived from Solanum lycopersicum L. 'Oregon Spring' grown under organic and conventional fertilizer conditions to evaluate the following hypotheses. 1. Organic soil fertilizer management results in greater allocation of photosynthetically derived resources to the synthesis of secondary metabolites than to plant growth, and 2. Genes involved in changes in the accumulation of phytonutrients under organic fertilizer regime will exhibit differential expression, and that the growth under different fertilizer treatments will elicit a differential response from the tomato genome. Both these hypotheses were supported, suggesting an adjustment of the metabolic and genomic activity of the plant in response to different fertilizers. Organic fertilizer treatment showed an activation of photoinhibitory processes through differential activation of nitrogen transport and assimilation genes resulting in higher accumulation of phytonutrients. This information can be used to identify alleles for breeding crops that allow for efficient utilization of organic inputs.

Publisher Correction: Dibutyl phthalate induced testicular dysgenesis originates after seminiferous cord formation in rats.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Long-term exposure to chemicals in sewage sludge fertilizer alters liver lipid content in females and cancer marker expression in males.

BACKGROUND: The increased incidence of diseases, including metabolic syndrome and infertility, may be related to exposure to the mixture of chemicals, which are ubiquitous in the modern environment (environmental chemicals, ECs). Xeno-detoxification occurs within the liver which is also the source of many plasma proteins and growth factors and plays an important role in the regulation of homeostasis. OBJECTIVES: The objective of this study was to investigate the effects of ECs on aspects of liver function, in a well characterized ovine model of exposure to a real-life EC mixture. METHODS: Four groups of sheep (n = 10-12/sex/treatment) were maintained long-term on control or sewage sludge-fertilized pastures: from conception to culling at 19 months of age in females and from conception to 7 months of age and thereafter in control plots until culling at 19 months of age in males. Environmental chemicals were measured in sheep livers and RNA and protein extracts were assessed for exposure markers. Liver proteins were resolved using 2D differential in-gel electrophoresis and differentially expressed protein spots were identified by liquid chromatography/tandem mass spectroscopy. RESULTS: Higher levels of polycyclic aromatic hydrocarbons (PAHs) and lower levels of polychlorinated biphenyls (PCBs) in the livers of control males compared to control females indicated sexually dimorphic EC body burdens. Increased levels of the PAHs Benzo[a]anthracene and chrysene and reduced levels of PCB 153 and PCB 180 were observed in the livers of continuously exposed females. EC exposure affected xenobiotic and detoxification responses and the liver proteome in both sexes and included major plasma-secreted and blood proteins, and metabolic enzymes whose pathway analysis predicted dysregulation of cancer-related pathways and altered lipid dynamics. The latter were confirmed by a reduction in total lipids in female livers and up-regulation of cancer-related transcript markers in male livers respectively by sewage sludge exposure. CONCLUSIONS: Our results demonstrate that chronic exposure to ECs causes major physiological changes in the liver, likely to affect multiple systems in the body and which may predispose individuals to increased disease risks.

Nodal Signaling Regulates Germ Cell Development and Establishment of Seminiferous Cords in the Human Fetal Testis.

Disruption of human fetal testis development is widely accepted to underlie testicular germ cell cancer (TGCC) origin and additional disorders within testicular dysgenesis syndrome (TDS). However, the mechanisms for the development of testicular dysgenesis in humans are unclear. We used ex vivo culture and xenograft approaches to investigate the importance of Nodal and Activin signaling in human fetal testis development. Inhibition of Nodal, and to some extent Activin, signaling disrupted seminiferous cord formation, abolished AMH expression, reduced androgen secretion, and decreased gonocyte numbers. Subsequent xenografting of testicular tissue rescued the disruptive effects on seminiferous cords and somatic cells but not germ cell effects. Stimulation of Nodal signaling increased the number of germ cells expressing pluripotency factors, and these persisted after xenografting. Our findings suggest a key role for Nodal signaling in the regulation of gonocyte differentiation and early human testis development with implications for the understanding of TGCC and TDS origin.

DMRT1 repression using a novel approach to genetic manipulation induces testicular dysgenesis in human fetal gonads.

STUDY QUESTION: Does loss of DMRT1 in human fetal testis alter testicular development and result in testicular dysgenesis? SUMMARY ANSWER: DMRT1 repression in human fetal testis alters the expression of key testicular and ovarian determining genes, and leads to focal testicular dysgenesis. WHAT IS KNOWN ALREADY: Testicular dysgenesis syndrome (TDS) is associated with common testicular disorders in young men, but its etiology is unknown. DMRT1 has been shown to play a role in the regulation of sex differentiation in the vertebrate gonad. Downregulation of DMRT1 in male mice results in trans-differentiation of Sertoli cells into granulosa (FOXL2+) cells resulting in an ovarian gonadal phenotype. STUDY DESIGN, SIZE, DURATION: To determine the effect of DMRT1 repression on human fetal testes, we developed a novel system for genetic manipulation, which utilizes a Lentivral delivered miRNA during short-term in vitro culture (2 weeks). A long-term (4-6 weeks) ex vivo xenograft model was used to determine the subsequent effects of DMRT1 repression on testicular development and maintenance. We included first and second-trimester testis tissue (8-20 weeks gestation; n = 12) in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human fetal testes were cultured in vitro and exposed to either of two DMRT1 miRNAs (miR536, miR641), or to scrambled control miRNA, for 24 h. This was followed by a further 14 days of culture (n = 3-4), or xenografting (n = 5) into immunocompromised mice for 4-6 weeks. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence and quantitative RT-PCR. Endpoints included histological evaluation of seminiferous cord integrity, mRNA expression of testicular, ovarian and germ cell genes, and assessment of cell number and protein expression for proliferation, apoptosis and pluripotency factors. Statistical analysis was performed using a linear mixed effect model. MAIN RESULTS AND THE ROLE OF CHANCE: DMRT1 repression (miR536/miR641) resulted in a loss of DMRT1 protein expression in a sub-population of Sertoli cells of first trimester (8-11 weeks gestation) human fetal testis; however, this did not affect the completion of seminiferous cord formation or morphological appearance. In second-trimester testis (12-20 weeks gestation), DMRT1 repression (miR536/miR641) resulted in disruption of seminiferous cords with absence of DMRT1 protein expression in Sertoli (SOX9+) cells. No differences in proliferation (Ki67+) were observed and apoptotic cells (CC3+) were rare. Expression of the Sertoli cell associated gene, SOX8, was significantly reduced (miR536, 34% reduction, P = 0.031; miR641 36% reduction, P = 0.026), whilst SOX9 expression was unaffected. Changes in expression of AMH (miR536, 100% increase, P = 0.033), CYP26B1 (miR641, 38% reduction, P = 0.05) and PTGDS (miR642, 30% reduction, P = 0.0076) were also observed. Amongst granulosa cell associated genes, there was a significant downregulation in R-spondin 1 expression (miR536, 76% reduction, P < 0.0001; miR641, 49% reduction, P = 0.046); however, there were no changes in expression of the granulosa cell marker, FOXL2. Analysis of germ cell associated genes demonstrated a significant increase in the expression of the pluripotency gene OCT4 (miR536, 233%, P < 0.001). We used the xenograft system to investigate the longer-term effects of seminiferous cord disruption via DMRT1 repression. As was evident in vitro for second-trimester samples, DMRT1 repression resulted in focal testicular dysgenesis similar to that described in adults with TDS. These dysgenetic areas were devoid of germ cells, whilst expression of FOXL2 within the dysgenetic areas, indicated trans-differentiation from a male (Sertoli cell) to female (granulosa cell) phenotype. LIMITATIONS, REASONS FOR CAUTION: Human fetal testis tissue is a limited resource; however, we were able to demonstrate significant effects of DMRT1 repression on the expression of germ and somatic cell genes, in addition to the induction of focal testicular dysgenesis, using these limited samples. In vitro culture may not reflect all aspects of human fetal testis development and function; however, the concurrent use of the xenograft model which represents a more physiological system supports the validity of the in vitro findings. WIDER IMPLICATIONS OF THE FINDINGS: Our findings have important implications for understanding the role of DMRT1 in human testis development and in the origin of testicular dysgenesis. In addition, we provide validation of a novel system that can be used to determine the effects of repression of genes that have been implicated in gonadal development and associated human reproductive disorders. STUDY FUNDING/COMPETING INTEREST(S): This project was funded by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) awarded to RTM. LBS was supported by MRC Programme Grant MR/N002970/1. RAA was supported by MRC Programme Grant G1100357/1. RMS was supported by MRC Programme Grant G33253. This work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. The funding bodies had no input into the conduct of the research or the production of this manuscript. The authors have declared no conflicts of interest.

Effects of Exposure to Acetaminophen and Ibuprofen on Fetal Germ Cell Development in Both Sexes in Rodent and Human Using Multiple Experimental Systems.

BACKGROUND: Analgesic exposure during pregnancy may affect aspects of fetal gonadal development that are targeted by endocrine disruptors. OBJECTIVES: We investigated whether therapeutically relevant doses of acetaminophen and ibuprofen affect germ cell (GC) development in human fetal testes/ovaries using in vitro and xenograft approaches. METHODS: First-trimester human fetal testes/ovaries were cultured and exposed to acetaminophen or ibuprofen (7 d). Second-trimester human fetal testes were xenografted into mice and exposed to acetaminophen (1 or 7 d), or ibuprofen (7 d). To determine mechanism of action, a human GC tumor­derived cell line (NTera2) exhibiting fetal GC characteristics was used in addition to in vitro and in vivo rat models. RESULTS AND DISCUSSION: Gonocyte (TFAP2C+) number was reduced relative to controls in first-trimester human fetal testes exposed in vitro to acetaminophen (-28%) or ibuprofen (-22%) and also in ovaries exposed to acetaminophen (-43%) or ibuprofen (-49%). Acetaminophen exposure reduced gonocyte number by 17% and 30% in xenografted second-trimester human fetal testes after treatment of host mice for 1 or 7 d, respectively. NTera2 cell number was reduced following exposure to either analgesic or prostaglandin E2 (PGE2) receptor antagonists, whereas PGE2 agonists prevented acetaminophen-induced reduction in NTera2 cell number. Expression of GC pluripotency genes, and genes that regulate DNA/histone methylation, also differed from controls following analgesic and PGE2 receptor antagonist exposures. Gene expression changes were observed in rat fetal testis/ovary cultures and after in vivo acetaminophen exposure of pregnant rats. For example, expression of the epigenetic regulator TET1, was increased following exposure to acetaminophen in human NTera2 cells, rat fetal testis/ovary cultures, and in fetal testes and ovaries after in vivo exposure of pregnant rats, indicating translatability across experimental models and species. CONCLUSIONS: Our results demonstrate evidence of PGE2-mediated effects of acetaminophen and ibuprofen on GC/NTera2 cells, which raises concerns about analgesic use during human pregnancy that warrant further investigation. https://doi.org/10.1289/EHP2307.

'Man Up': the importance and strategy for placing male reproductive health centre stage in the political and research agenda.

Approximately 1 in 20 young men today have sperm counts low enough to impair fertility, whereas this may not have been the case historically. The cause(s) of such a decline in male reproductive health is unknown, despite it being a global health issue. Concomitantly, little progress has been made in answering fundamental questions in andrology or in developing new diagnostic tools or alternative management strategies to ICSI in infertile men. We advocate formulation of a detailed roadmap for male reproductive health to facilitate development of a research agenda that highlights the present unmet needs and key unanswered questions, and seeks to deliver effective funding and investment to address them. This vision we term 'a Male Reproductive Health Ecosystem'.

Programmed for sex: Nutrition-reproduction relationships from an inter-generational perspective.

Reproduction is our biological reason for being. Our physiology has been shaped via countless millennia of evolution with this one purpose in mind, so that at birth we are 'programmed for sex', although this will not kick-start functionally until puberty. Our development from an early embryo is focused on making us fit to reproduce and is intimately connected to nutrition and energy stores. Fluctuations in food supply has probably been a key evolutionary shaper of the reproductive process, and this review hypothesizes that we have developed rapid, non-genomic adaptive mechanisms to such fluctuations to better fit offspring to their perceived (nutritional) environment, thus giving them a reproductive advantage. There is abundant evidence for this notion from 'fetal programming' studies and from experimental 'inter-generational' studies involving manipulation of parental (especially paternal) diet and then examining metabolic changes in resulting offspring. It is argued that the epigenetic reprogramming of germ cells that occurs during fetal life, after fertilisation and during gametogenesis provides opportunities for sensing of the (nutritional) environment so as to affect adaptive epigenetic changes to alter offspring metabolic function. In this regard, there may be adverse effects of a modern Western diet, perhaps because it is deficient in plant-derived factors that are proven to be capable of altering the epigenome, folate being a prime example; we have evolved in tune with such factors. Therefore, parental and even grandparental diets may have consequences for health of future generations, but how important this might be and the precise epigenetic mechanisms involved are unknown.

Low-dose tamoxifen treatment in juvenile males has long-term adverse effects on the reproductive system: implications for inducible transgenics.

The tamoxifen-inducible Cre system is a popular transgenic method for controlling the induction of recombination by Cre at a specific time and in a specific cell type. However, tamoxifen is not an inert inducer of recombination, but an established endocrine disruptor with mixed agonist/antagonist activity acting via endogenous estrogen receptors. Such potentially confounding effects should be controlled for, but >40% of publications that have used tamoxifen to generate conditional knockouts have not reported even the minimum appropriate controls. To highlight the importance of this issue, the present study investigated the long-term impacts of different doses of a single systemic tamoxifen injection on the testis and the wider endocrine system. We found that a single dose of tamoxifen less than 10% of the mean dose used for recombination induction, caused adverse effects to the testis and to the reproductive endocrine system that persisted long-term. These data raise significant concerns about the widespread use of tamoxifen induction of recombination, and highlight the importance of including appropriate controls in all pathophysiological studies using this means of induction.

Dibutyl phthalate induced testicular dysgenesis originates after seminiferous cord formation in rats.

Administration of dibutyl phthalate (DBP) to pregnant rats causes reproductive disorders in male offspring, resulting from suppression of intratesticular testosterone, and is used as a model for human testicular dysgenesis syndrome (TDS). DBP exposure in pregnancy induces focal dysgenetic areas in fetal testes that appear between e19.5-e21.5, manifesting as focal aggregation of Leydig cells and ectopic Sertoli cells (SC). Our aim was to identify the origins of the ectopic SC. Time-mated female rats were administered 750 mg/kg/day DBP in three different time windows: full window (FW; e13.5-e20.5), masculinisation programming window (MPW; e15.5-e18.5), late window (LW; e19.5-e20.5). We show that DBP-MPW treatment produces more extensive and severe dysgenetic areas, with more ectopic SC and germ cells (GC) than DBP-FW treatment; DBP-LW induces no dysgenesis. Our findings demonstrate that ectopic SC do not differentiate de novo, but result from rupture of normally formed seminiferous cords beyond e20.5. The more severe testis dysgenesis in DBP-MPW animals may result from the presence of basally migrating GC and a weakened basal lamina, whereas GC migration was minimal in DBP-FW animals. Our findings provide the first evidence for how testicular dysgenesis can result after normal testis differentiation/development and may be relevant to understanding TDS in human patients.