Adenomyotic Lesions Are Induced in the Mouse Uterus after Exposure to NSAID and EE2 Mixtures at Environmental Doses.

The aim of this study was to assess the long-term effect of exposure to environmentally relevant doses of non-steroidal anti-inflammatory drugs (NSAIDs; ibuprofen, and diclofenac) and 17ß-ethinylestradiol (EE2) on the mouse uterus. NSAID-EE2 mixtures were administered in the drinking water from gestational day 8 until 8 weeks post-birth (i.e., during embryo development, lactation, puberty, and sexual maturity). The incidence of adenomyosis lesions (presence of endometrial glands in the inner myometrium) increased up to 60% in the uterus of 8-week-old exposed females (F1) and to 85% in F2 females (exposed father). Histological analysis revealed aberrant proliferation and apoptosis, vacuolization of epithelial cells, and increased incidence of abnormal glands in the luminal and glandular epithelium in F1 and F2 uteri. Moreover, myofibroblast proportion (alpha-smooth muscle actin (α-SMA) expression analysis) and collagen expression (Picrosirius red stain; a fibrosis hallmark) were increased in F1 and F2 endometrium. Connexin-43 was aberrantly distributed in the endometrial stroma and glands of F1 and F2 uteri. Conversely, uterine 17ß-estradiol and progesterone levels were not affected in F1 and F2 females. These findings demonstrated that in mice, chronic exposure to NSAID and EE2 mixtures at environmental doses intergenerationally affects uterine physiology, particularly the endometrium. It may serve as a model to study the pathophysiology of human adenomyosis.

Intergenerational effects on fertility in male and female mice after chronic exposure to environmental doses of NSAIDs and 17α-ethinylestradiol mixtures.

Non-steroidal anti-inflammatory drugs (NSAIDs) and 17α-ethinylestradiol (EE2) are extensively used in human and veterinary medicine. Due to their partial removal by wastewater treatment plants, they are frequent environmental contaminants, particularly in drinking water. Here, we investigated the adverse outcomes of chronic exposure to mixtures of NSAIDs (ibuprofen, 2hydroxy-ibuprofen, diclofenac) and EE2 at two environmentally relevant doses in drinking water, on the reproductive organ development and fertility in F1-exposed male and female mice and in their F2 offspring. In male and female F1 mice, which were exposed to these mixtures, reproductive organ maturation, estrous cyclicity, and spermiogenesis were altered. These defects were observed also in F2 animals, in addition to some specific sperm parameter alterations in F2 males. Transcriptomic analysis revealed significant changes in gene expression patterns and associated pathways implicated in testis and ovarian physiology. Chronic exposure of mice to NSAID and EE2 mixtures at environmental doses intergenerationally affected male and female fertility (i.e. total number of pups and time between litters). Our study provides new insights into the adverse effects of these pharmaceuticals on the reproductive health and will facilitate the implementation of a future regulatory environmental risk assessment of NSAIDs and EE2 for human health.

Cocktails of NSAIDs and 17α Ethinylestradiol at Environmentally Relevant Doses in Drinking Water Alter Puberty Onset in Mice Intergenerationally.

Non-steroidal anti-inflammatory drugs (NSAIDs) and 17α-ethinyl-estradiol (EE2) are among the most relevant endocrine-disrupting pharmaceuticals found in the environment, particularly in surface and drinking water due to their incomplete removal via wastewater treatment plants. Exposure of pregnant mice to NSAID therapeutic doses during the sex determination period has a negative impact on gonadal development and fertility in adults; however, the effects of their chronic exposure at lower doses are unknown. In this study, we investigated the impact of chronic exposure to a mixture containing ibuprofen, 2hydroxy-ibuprofen, diclofenac, and EE2 at two environmentally relevant doses (added to the drinking water from fetal life until puberty) on the reproductive tract in F1 exposed mice and their F2 offspring. In F1 animals, exposure delayed male puberty and accelerated female puberty. In post-pubertal F1 testes and ovaries, differentiation/maturation of the different gonad cell types was altered, and some of these modifications were observed also in the non-exposed F2 generation. Transcriptomic analysis of post-pubertal testes and ovaries of F1 (exposed) and F2 animals revealed significant changes in gene expression profiles and enriched pathways, particularly the inflammasome, metabolism and extracellular matrix pathways, compared with controls (non-exposed). This suggested that exposure to these drug cocktails has an intergenerational impact. The identified Adverse Outcome Pathway (AOP) networks for NSAIDs and EE2, at doses that are relevant to everyday human exposure, will improve the AOP network of the human reproductive system development concerning endocrine disruptor chemicals. It may serve to identify other putative endocrine disruptors for mammalian species based on the expression of biomarkers.

TRIM28-dependent SUMOylation protects the adult ovary from activation of the testicular pathway.

Gonadal sexual fate in mammals is determined during embryonic development and must be actively maintained in adulthood. In the mouse ovary, oestrogen receptors and FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying their protective effect is unknown. Here, we show that TRIM28 is required to prevent female-to-male sex reversal of the mouse ovary after birth. We found that upon loss of Trim28, ovarian granulosa cells transdifferentiate to Sertoli cells through an intermediate cell type, different from gonadal embryonic progenitors. TRIM28 is recruited on chromatin in the proximity of FOXL2 to maintain the ovarian pathway and to repress testicular-specific genes. The role of TRIM28 in ovarian maintenance depends on its E3-SUMO ligase activity that regulates the sex-specific SUMOylation profile of ovarian-specific genes. Our study identifies TRIM28 as a key factor in protecting the adult ovary from the testicular pathway.

Using Experimental Models to Decipher the Effects of Acetaminophen and NSAIDs on Reproductive Development and Health.

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin (acetylsalicylic acid), diclofenac and ibuprofen (IBU), and analgesic drugs, such as acetaminophen (APAP, or paracetamol), are widely used to treat inflammation and pain. APAP and IBU are over-the-counter drugs and are among the most commonly taken drugs in the first trimester of pregnancy, even in combination. Furthermore, these drugs and their metabolites are released in the environment, and can be frequently detected in wastewater, surface water, and importantly in drinking water. Although their environmental concentrations are much lower than the therapeutics doses, this suggests an uncontrolled low-dose exposure of the general population, including pregnant women and young children, two particularly at risk populations. Epidemiological studies show that exposure to these molecules in the first and second trimester of gestation can favor genital malformations in new-born boys. To investigate the cellular, molecular and mechanistic effects of exposure to these molecules, ex vivo studies with human or rodent gonadal explants and in vivo experiments in rodents have been performed in the past years. This review recapitulates recent data obtained in rodent models after in utero or postnatal exposure to these drugs. The first part of this review discusses the mechanisms by which NSAIDs and analgesics may impair gonadal development and maturation, puberty development, sex hormone production, maturation and function of adult organs, and ultimately fertility in the exposed animals and their offspring. Like other endocrine disruptors, NSAIDs and APAP interfere with endocrine gland function and may have inter/transgenerational adverse effects. Particularly, they may target germ cells, resulting in reduced quality of male and female gametes, and decreased fertility of exposed individuals and their descendants. Then, this review discusses the effects of exposure to a single drug (APAP, aspirin, or IBU) or to combinations of drugs during early embryogenesis, and the consequences on postnatal gonadal development and adult reproductive health. Altogether, these data may increase medical and public awareness about these reproductive health concerns, particularly in women of childbearing age, pregnant women, and parents of young children.

In the mouse, prostaglandin D2 signalling protects the endometrium against adenomyosis.

Adenomyosis is characterised by epithelial gland and mesenchymal stroma invasion of the uterine myometrium. Adenomyosis is an oestrogen-dependent gynaecological disease in which a number of factors, such as inflammatory molecules, prostaglandins (PGs), angiogenic factors, cell proliferation and extracellular matrix remodelling proteins, also play a role as key disease mediators. In this study, we used mice lacking both lipocalin and hematopoietic-PG D synthase (L- and H-Pgds) genes in which PGD2 is not produced to elucidate PGD2 roles in the uterus. Gene expression studied by real-time PCR and hormone dosages performed by ELISA or liquid chromatography tandem mass spectroscopy in mouse uterus samples showed that components of the PGD2 signalling pathway, both PGDS and PGD2-receptors, are expressed in the mouse endometrium throughout the oestrus cycle with some differences among uterine compartments. We showed that PGE2 production and the steroidogenic pathway are dysregulated in the absence of PGD2. Histological analysis of L/H-Pgds-/- uteri, and immunohistochemistry and immunofluorescence analyses of proliferation (Ki67), endothelial cell (CD31), epithelial cell (pan-cytokeratin), myofibroblast (α-SMA) and mesenchymal cell (vimentin) markers, identify that 6-month-old L/H-Pgds-/- animals developed adenomyotic lesions, and that disease severity increased with age. In conclusion, this study suggests that the PGD2 pathway has major roles in the uterus by protecting the endometrium against adenomyosis development. Additional experiments, using for instance transcriptomic approaches, are necessary to fully determine the molecular mechanisms that lead to adenomyosis in L/H-Pgds-/- mice and to confirm whether this strain is an appropriate model for studying the human disease.

In utero exposure to acetaminophen and ibuprofen leads to intergenerational accelerated reproductive aging in female mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs) and analgesic drugs, such as acetaminophen (APAP), are frequently taken during pregnancy, even in combination. However, they can favour genital malformations in newborn boys and reproductive disorders in adults. Conversely, the consequences on postnatal ovarian development and female reproductive health after in utero exposure are unknown. Here, we found that in mice, in utero exposure to therapeutic doses of the APAP-ibuprofen combination during sex determination led to delayed meiosis entry and progression in female F1 embryonic germ cells. Consequently, follicular activation was reduced in postnatal ovaries through the AKT/FOXO3 pathway, leading in F2 animals to subfertility, accelerated ovarian aging with abnormal corpus luteum persistence, due to decreased apoptosis and increased AKT-mediated luteal cell survival. Our study suggests that administration of these drugs during the critical period of sex determination could lead in humans to adverse effects that might be passed to the offspring.

Intergenerational effects on mouse sperm quality after in utero exposure to acetaminophen and ibuprofen.

Nonsteroidal antiinflammatory drugs and analgesic drugs, such as N-acetyl- p-aminophenol (APAP; acetaminophen, paracetamol), are widely used by pregnant women. Accumulating evidence has indicated that these molecules can favor genital malformations in newborn boys and reproductive disorders in adults. However, the consequences on postnatal testis development and adult reproductive health after exposure during early embryogenesis are still unknown. Using the mouse model, we show that in utero exposure to therapeutic doses of the widely used APAP-ibuprofen combination during the sex determination period leads to early differentiation and decreased proliferation of male embryonic germ cells, and early 5-methylcytosine and extracellular matrix protein deposition in 13.5 d postcoitum exposed testes. Consequently, in postnatal testes, Sertoli-cell maturation is delayed, the Leydig-cell compartment is hyperplasic, and the spermatogonia A pool is decreased. This results in a reduced production of testosterone and in epididymal sperm parameter defects. We observed a reduced sperm count (19%) in utero-exposed (F0) adult males and also a reduced sperm motility (40%) in their offspring (F1) when both parents were exposed, which leads to subfertility among the 6 mo old F1 animals. Our study suggests that the use of these drugs during the critical period of sex determination affects the germ-line development and leads to adverse effects that could be passed to the offspring.-Rossitto, M., Marchive, C., Pruvost, A., Sellem, E., Ghettas, A., Badiou, S., Sutra, T., Poulat, F., Philibert, P., Boizet-Bonhoure, B. Intergenerational effects on mouse sperm quality after in utero exposure to acetaminophen and ibuprofen.

The Mouse Microbiome Is Required for Sex-Specific Diurnal Rhythms of Gene Expression and Metabolism.

The circadian clock and associated feeding rhythms have a profound impact on metabolism and the gut microbiome. To what extent microbiota reciprocally affect daily rhythms of physiology in the host remains elusive. Here, we analyzed transcriptome and metabolome profiles of male and female germ-free mice. While mRNA expression of circadian clock genes revealed subtle changes in liver, intestine, and white adipose tissue, germ-free mice showed considerably altered expression of genes associated with rhythmic physiology. Strikingly, the absence of the microbiome attenuated liver sexual dimorphism and sex-specific rhythmicity. The resulting feminization of male and masculinization of female germ-free animals is likely caused by altered sexual development and growth hormone secretion, associated with differential activation of xenobiotic receptors. This defines a novel mechanism by which the microbiome regulates host metabolism.

Microtubule polyglutamylation and acetylation drive microtubule dynamics critical for platelet formation.

BACKGROUND: Upon maturation in the bone marrow, polyploid megakaryocytes elongate very long and thin cytoplasmic branches called proplatelets. Proplatelets enter the sinusoids blood vessels in which platelets are ultimately released. Microtubule dynamics, bundling, sliding, and coiling, drive these dramatic morphological changes whose regulation remains poorly understood. Microtubule properties are defined by tubulin isotype composition and post-translational modification patterns. It remains unknown whether microtubule post-translational modifications occur in proplatelets and if so, whether they contribute to platelet formation. RESULTS: Here, we show that in proplatelets from mouse megakaryocytes, microtubules are both acetylated and polyglutamylated. To bypass the difficulties of working with differentiating megakaryocytes, we used a cell model that allowed us to test the functions of these modifications. First, we show that α2bß3integrin signaling in D723H cells is sufficient to induce ß1tubulin expression and recapitulate the specific microtubule behaviors observed during proplatelet elongation and platelet release. Using this model, we found that microtubule acetylation and polyglutamylation occur with different spatio-temporal patterns. We demonstrate that microtubule acetylation, polyglutamylation, and ß1tubulin expression are mandatory for proplatelet-like elongation, swelling formation, and cytoplast severing. We discuss the functional importance of polyglutamylation of ß1tubulin-containing microtubules for their efficient bundling and coiling during platelet formation. CONCLUSIONS: We characterized and validated a powerful cell model to address microtubule behavior in mature megakaryocytes, which allowed us to demonstrate the functional importance of microtubule acetylation and polyglutamylation for platelet release. Furthermore, we bring evidence of a link between the expression of a specific tubulin isotype, the occurrence of microtubule post-translational modifications, and the acquisition of specific microtubule behaviors. Thus, our findings could widen the current view of the regulation of microtubule behavior in cells such as osteoclasts, spermatozoa, and neurons, which express distinct tubulin isotypes and display specific microtubule activities during differentiation.

In mammalian foetal testes, SOX9 regulates expression of its target genes by binding to genomic regions with conserved signatures.

In mammalian embryonic gonads, SOX9 is required for the determination of Sertoli cells that orchestrate testis morphogenesis. To identify genetic networks directly regulated by SOX9, we combined analysis of SOX9-bound chromatin regions from murine and bovine foetal testes with sequencing of RNA samples from mouse testes lacking Sox9. We found that SOX9 controls a conserved genetic programme that involves most of the sex-determining genes. In foetal testes, SOX9 modulates both transcription and directly or indirectly sex-specific differential splicing of its target genes through binding to genomic regions with sequence motifs that are conserved among mammals and that we called 'Sertoli Cell Signature' (SCS). The SCS is characterized by a precise organization of binding motifs for the Sertoli cell reprogramming factors SOX9, GATA4 and DMRT1. As SOX9 biological role in mammalian gonads is to determine Sertoli cells, we correlated this genomic signature with the presence of SOX9 on chromatin in foetal testes, therefore equating this signature to a genomic bar code of the fate of foetal Sertoli cells. Starting from the hypothesis that nuclear factors that bind to genomic regions with SCS could functionally interact with SOX9, we identified TRIM28 as a new SOX9 partner in foetal testes.

Molecular events and signalling pathways of male germ cell differentiation in mouse.

Germ cells, the precursors of gametes, represent a unique cell lineage that is able to differentiate into spermatozoa or oocytes depending on the chromosomal sex of the organism. In the mammalian embryonic gonad, commitment to oogenesis involves pre-meiotic DNA replication and entry into the first meiotic division; whereas, commitment to spermatogenesis involves inhibition of meiotic initiation, suppression of pluripotency, mitotic arrest and expression of specific markers that will control the development of the male germ cells. The crucial decision made by the germ line to commit to either a male or a female fate has been partially explained by genetic and ex vivo studies in mice which have implicated a complex network of regulatory genes, numerous factors and pathways. Besides the reproductive failure that may follow a deregulation of this complex network, the germ cells may, in view of their proliferative and pluripotent nature, act as precursors of potential malignant transformation and as putative targets for exogenous environmental compounds. Our review summarizes and discusses recent developments that have improved our understanding on how germ cell precursors are committed to a male or a female cell fate in the mouse gonad.

Multiple roles of the prostaglandin D2 signaling pathway in reproduction.

Prostaglandins signaling molecules are involved in numerous physiological processes. They are produced by several enzyme-limited reactions upon fatty acids, which are catalyzed by two cyclooxygenases and prostaglandin synthases. In particular, the prostaglandins E2 (PGE2), D2 (PGD2), and F2 (PGF2 α) have been shown to be involved in female reproductive mechanisms. Furthermore, widespread expression of lipocalin- and hematopoietic-PGD2 synthases in the male reproductive tract supports the purported roles of PGD2 in the development of both embryonic and adult testes, sperm maturation, and spermatogenesis. In this review, we summarize the putative roles of PGD2 signaling and the roles of both PGD2 synthases in testicular formation and function. We review the data reporting the involvement of PGD2 signaling in the differentiation of Sertoli and germ cells of the embryonic testis. Furthermore, we discuss the roles of lipocalin-PGD2 synthase in steroidogenesis and spermatogenesis, in terms of lipid molecule transport and PGD2 production. Finally, we discuss the hypothesis that PGD2 signaling may be affected in certain reproductive diseases, such as infertility, cryptorchidism, and testicular cancer.

Prostaglandin D2 synthase/GPR44: a signaling axis in PNS myelination.

Neuregulin 1 type III is processed following regulated intramembrane proteolysis, which allows communication from the plasma membrane to the nucleus. We found that the intracellular domain of neuregulin 1 type III upregulated the prostaglandin D2 synthase (L-pgds, also known as Ptgds) gene, which, together with the G protein-coupled receptor Gpr44, forms a previously unknown pathway in PNS myelination. Neuronal L-PGDS is secreted and produces the PGD2 prostanoid, a ligand of Gpr44. We found that mice lacking L-PGDS were hypomyelinated. Consistent with this, specific inhibition of L-PGDS activity impaired in vitro myelination and caused myelin damage. Furthermore, in vivo ablation and in vitro knockdown of glial Gpr44 impaired myelination. Finally, we identified Nfatc4, a key transcription factor for myelination, as one of the downstream effectors of PGD2 activity in Schwann cells. Thus, L-PGDS and Gpr44 are previously unknown components of an axo-glial interaction that controls PNS myelination and possibly myelin maintenance.

Anatomical and molecular analyses of XY ovaries from the African pygmy mouse Mus minutoides.

The African pygmy mouse Mus minutoides is characterized by the presence of a high proportion of fertile XY females in natural populations. This species displays 2 morphologically different X chromosomes: the ancestral X and a shorter one designated as X*, feminizing the X*Y individuals. This strongly suggests that in the presence of an X* chromosome, the male differentiation program is not activated despite a functional Y chromosome. In this study, we compared the histology of the adult ovaries of the 3 female genotypes (XX, XX* and X*Y) and investigated the expression of some of the main genes involved in male and female differentiation. We found that X*Y gonads display a typical ovarian structure without any testicular organization. Moreover, the ovarian somatic marker FOXL2 is detected in X*Y follicle cells and exhibits the same pattern as in XX and XX* ovaries, whereas SOX9 and DMRT1 are absent at all stages of follicular differentiation. However, surprisingly, X*Y ovaries display a higher level of Sry transcripts compared to testes. Our findings confirm the complete sex reversal in X*Y individuals with no apparent sign of masculinization, providing an attractive model to unravel new gene interactions involved in the mammalian sex determination system.

Prostaglandin D2 acts through the Dp2 receptor to influence male germ cell differentiation in the foetal mouse testis.

Through intercellular signalling, the somatic compartment of the foetal testis is able to program primordial germ cells to undergo spermatogenesis. Fibroblast growth factor 9 and several members of the transforming growth factor ß superfamily are involved in this process in the foetal testis, counteracting the induction of meiosis by retinoic acid and activating germinal mitotic arrest. Here, using in vitro and in vivo approaches, we show that prostaglandin D2 (PGD2), which is produced through both L-Pgds and H-Pgds enzymatic activities in the somatic and germ cell compartments of the foetal testis, plays a role in mitotic arrest in male germ cells by activating the expression and nuclear localization of the CDK inhibitor p21(Cip1) and by repressing pluripotency markers. We show that PGD2 acts through its Dp2 receptor, at least in part through direct effects in germ cells, and contributes to the proper differentiation of male germ cells through the upregulation of the master gene Nanos2. Our data identify PGD2 signalling as an early pathway that acts in both paracrine and autocrine manners, and contributes to the differentiation of germ cells in the foetal testis.

Unilateral cryptorchidism in mice mutant for Ptgds.

The pathophysiology of cryptorchidism, abnormal testicular descent, remains poorly understood. In this study, we show that both heterozygous and homozygous mice deficient for lipocalin-type prostaglandin D(2) (PGD(2) ) synthase (Ptgds) presented unilateral cryptorchidism affecting the second phase of testicular descent in 16% and 24% of cases, respectively. The adult cryptorchid testes show an increase in spermatogonia apoptosis along with a global decrease in the tubule size parameters, whereas the gubernaculum of newborn mutants present some histological abnormalities. Disruption of the inguinoscrotal phase did not present impairment of the androgen pathway but rather a decrease in Rxfp2 mRNA expression in the gubernaculum. These observations led us to investigate the role of the PGD(2) signaling pathway in human testicular migration through PTGDS sequencing of DNA from 29 children with cryptorchidism. However, none of the investigated cases presented mutations in the PTGDS gene. Nevertheless, our results identify the PTGDS enzyme as a novel component in the cryptorchidism puzzle.

Hematopoietic prostaglandin D synthase (H-Pgds) is expressed in the early embryonic gonad and participates to the initial nuclear translocation of the SOX9 protein.

In mammals, the Prostaglandin D(2) (PGD(2) ) signaling pathway is involved in male gonadal development, regulating Sox9 gene expression and SOX9 protein subcellular localization through lipocalin prostaglandin D synthase (L-Pgds) activity. Nevertheless, because L-Pgds is downstream of Sox9, its expression cannot explain the initial nuclear translocation of the SOX9 protein. Here, we show that another source of PGD(2) , hematopoietic-Pgds (H-Pgds) enzyme is expressed in somatic and germ cells of the embryonic gonad of both sexes, as early as embryonic day (E) 10.5, before the onset of L-Pgds expression. Inhibition of H-Pgds activity by the specific HQL-79 inhibitor leads to impaired nuclear translocation of SOX9 protein in E11.5 Sertoli cells. Furthermore, analysis of H-Pgds(-/-) male embryonic gonads confirms abnormal subcellular localization of SOX9 protein at the E11.5 early stage of mouse testicular differentiation suggesting a role for H-Pgds-produced PGD(2) in the initial nuclear translocation of SOX9.

Inhibition of SRY-calmodulin complex formation induces ectopic expression of ovarian cell markers in developing XY gonads.

The transcription factor sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, because mutations in SRY cause disorders of sex development in XY individuals. During gonadal development, Sry in pre-Sertoli cells activates Sox9 gene transcription, committing the fate of the bipotential gonad to become a testis rather than an ovary. The high-mobility group domain of human SRY contains two independent nuclear localization signals, one bound by calmodulin (CaM) and the other by importin-ß. Although XY females carry SRY mutations in these nuclear localization signals that affect SRY nuclear import in transfected cells, it is not known whether these transport mechanisms are essential for gonadal development and sex determination. Here, we show that mouse Sry protein binds CaM and that a CaM antagonist reduces CaM binding, nuclear accumulation, and transcriptional activity of Sry in transfected cells. CaM antagonist treatment of cultured, sexually indifferent XY mouse fetal gonads led to reduced expression of the Sry target gene Sox9, defects in testicular cord formation, and ectopic expression of the ovarian markers Rspondin1 and forkhead box L2. These results indicate the importance of CaM for SRY nuclear import, transcriptional activity, testis differentiation, and sex determination.