David Møbjerg Kristensen

Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract

BACKGROUND The vitamin D receptor (VDR) is expressed in human testis, and vitamin D (VD) has been suggested to affect survival and function of mature spermatozoa. Indeed, VDR knockout mice and VD deficient rats show decreased sperm counts and low fertility. However, the cellular response to VD is complex, since it is not solely dependent on VDR expression, but also on cellular uptake of circulating VD and presence and activity of VD metabolizing enzymes. Expression of VD metabolizing enzymes has not previously been investigated in human testis and male reproductive tract. Therefore, we performed a comprehensive analysis of the expression of VDR, VD activating (CYP2R1, CYP27A1, CYP27B1) and inactivating (CYP24A1) enzymes in the testis, epididymis, seminal vesicle (SV), prostate and spermatozoa. METHODS Tissue samples were obtained after orchiectomy (testis n = 13; epididymis n = 7), prostatectomy (prostate n = 5 and SVs n = 3) and semen samples obtained after ejaculation (n = 13). mRNA was detected with RT-PCR and expression of proteins was determined by immunohistochemistry. RESULTS VDR and VD metabolizing enzymes were concomitantly expressed in round and elongated spermatids, vesicles within the caput epididymis, and glandular epithelium of cauda epididymis, SV and prostate. The expression pattern in ejaculated spermatozoa varied, although, concomitant expression of VDR, CYP2R1, CYP27B1 and CYP24A1 was observed in neck and midpiece in a subpopulation of mature spermatozoa. CONCLUSION On the basis of the marked expression of VDR and the VD metabolizing enzymes in human testis, ejaculatory tract and mature spermatozoa, we suggest that VD is important for spermatogenesis and maturation of human spermatozoa.

Intrauterine exposure to mild analgesics is a risk factor for development of male reproductive disorders in human and rat

BACKGROUND More than half of pregnant women in the Western world report intake of mild analgesics, and some of these drugs have been associated with anti-androgenic effects in animal experiments. Intrauterine exposure to anti-androgens is suspected to contribute to the recent increase in male reproductive problems, and many of the anti-androgenic compounds are like the mild analgesics potent inhibitors of prostaglandin synthesis. Therefore, it appears imperative to further investigate the potential endocrine disrupting properties of mild analgesics. METHODS In a prospective birth cohort study, 2297 Danish and Finnish pregnant women completed a questionnaire and 491 of the Danish mothers participated in a telephone interview, reporting on their use of mild analgesics during pregnancy. The testicular position of newborns was assessed by trained paediatricians. In rats, the impact of mild analgesics on anogenital distance (AGD) after intrauterine exposure was examined together with the effect on ex vivo gestational day 14.5 testes. RESULTS In the Danish birth cohort, the use of mild analgesics was dose-dependently associated with congenital cryptorchidism. In particular, use during the second trimester increased the risk. This risk was further increased after the simultaneous use of different analgesics. The association was not found in the Finnish birth cohort. Intrauterine exposure of rats to paracetamol led to a reduction in the AGD and mild analgesics accordingly reduced testosterone production in ex vivo fetal rat testes. CONCLUSION There was an association between the timing and the duration of mild analgesic use during pregnancy and the risk of cryptorchidism. These findings were supported by anti-androgenic effects in rat models leading to impaired masculinization. Our results suggest that intrauterine exposure to mild analgesics is a risk factor for development of male reproductive disorders.

Presumed pluripotency markers UTF-1 and REX-1 are expressed in human adult testes and germ cell neoplasms

BACKGROUND UTF-1 and REX-1/ZFP42 are transcription factors involved in pluripotency. Because of phenotypic similarities between pluripotent embryonic stem cells and testicular germ cell tumours (TGCT) and the derivation of pluripotent cells from testes, we investigated the expression of UTF-1 and REX-1 during human gonadal development and in TGCT. METHODS Expression of UTF-1 and REX-1 was studied in 52 specimens from human gonadal development and in 86 samples from TGCT. RESULTS UTF-1 and REX-1 were expressed throughout male gonadal development. In the mature testis, UTF-1 was expressed in spermatogonia, whereas REX-1 was expressed in meiotic cells and, together with OCT-3/4, in primary oocytes. Both UTF-1 and REX-1 were expressed in testicular carcinoma in situ and in TGCT. Contrarily to REX-1, UTF-1 was expressed in all spermatocytic seminomas. CONCLUSIONS Unlike other pluripotency markers NANOG and OCT-3/4, UTF-1 and REX-1 are expressed throughout human testes development. The expression pattern indicated that UTF-1 plays a possible role in spermatogonial self-renewal, whereas expression of REX-1 in meiotic cells from both testes and ovary indicate a role in meiosis. UFT-1 and REX-1 are expressed in TGCT and the high abundance of UTF-1 in spermatocytic seminomas is consistent with the hypothesis that this tumour type originates from spermatogonia.

Paracetamol, Aspirin, and Indomethacin Induce Endocrine Disturbances in the Human Fetal Testis Capable of Interfering With Testicular Descent

CONTEXT Masculinization depends on the fetal testis. Exposure of the human fetus during pregnancy to paracetamol and/or to other mild analgesics is associated with an increased risk of cryptorchidism. OBJECTIVE We aimed to determine whether mild analgesics disrupted the morphology and endocrine function of the human testis. DESIGN We used an in vitro system based on the culture of human fetal testes exposed or not to paracetamol, its metabolite N-(4-hydroxyphenyl)-arachidonoylethanolamide (AM404), aspirin, indomethacin, and ketoconazole at 10(-4) to 10(-7) M. SETTING The study was conducted at the University of Rennes I. PATIENTS/PARTICIPANTS Human fetal testes were from pregnant women after induced abortion, between 7 and 12 weeks of gestation (GW). MAIN OUTCOME MEASURES Testosterone (RIA), anti-Müllerian hormone (ELISA), insulin-like factor 3 (RIA), and prostaglandin (PG) D2 and PGE2 (ELISA) were assayed in the medium. Testicular cells were counted using histology and image analysis. The possible nuclear receptor-mediated activities of the analgesics were investigated using reporter cell lines expressing estrogen, androgen, and peroxisome proliferator-activated γ receptors. RESULTS Indomethacin and aspirin stimulated testosterone production, particularly by the younger testes (8-9 GW vs 10-12 GW). Paracetamol, AM404, and ketoconazole decreased insulin-like factor 3 levels. Aspirin stimulated whereas ketoconazole inhibited AMH production. PGE2 levels were inhibited by paracetamol and aspirin in the 7 to 12 GW testes and by indomethacin but only in 7 to 9.86 GW testes. The inhibitory trends seen for PGD2 were not statistically significant. CONCLUSIONS Analgesics at concentrations relevant to human exposure cause endocrine disturbances in the fetal testis. We suggest that the fetal human testis displays slight critical age windows for sensitivity to direct exposure to aspirin, indomethacin, and paracetamol. The analgesic-induced inhibition of INSL3 may be the mechanism by which analgesics increase the risk of cryptorchidism. Greater caution is required concerning consumption of analgesics during pregnancy.

Origin of pluripotent germ cell tumours: The role of microenvironment during embryonic development

Carcinoma in situ (CIS) testis, known also as intratubular germ cell neoplasia, is the cancer stem cell from which the great majority of testicular germ cell derived tumours (TGCTs) of the testis arise. TGCTs can proliferate into morphologically homogeneous seminomas or can differentiate into virtually any type of tissue and form teratomas (non-seminomas). CIS cells display a close phenotypic similarity to fetal germ cells (primordial germ cells or gonocytes) suggesting an origin due to a developmental delay or arrest of differentiation of early germ cells. The pluripotency of these neoplasms has recently been explained by a close resemblance of their expression profile to that of embryonic inner cell mass cells studied in culture as embryonic stem cells, with high expression of transcription factors associated with pluripotency, such as NANOG and OCT3/4, as well as proteins found in several tissue specific stem cells, such as TFAP2C (AP-2gamma) or KIT. CIS and seminomas highly express a number of pre-meiotic germ cell specific genes, which are down-regulated during development to non-seminomas, while the expression of other embryonic markers, such as SOX2, is up-regulated. The mechanistic pathways and causative factors remain to be elucidated of both the initial transformation of fetal germ cells into CIS cells and the progression of CIS cells into an invasive tumour in the young adult. However, evidence supported by epidemiological studies indicate that disturbances in the hormonal microenvironment of the differentiating gonads may results in both the neoplasia and a host of other problems later in life, such as genital malformations, decreased spermatogenesis, and signs of hypogonadism.

Cytokine signalling in embryonic stem cells

Cytokines play a central role in maintaining self‐renewal in mouse embryonic stem (ES) cells through a member of the interleukin‐6 type cytokine family termed leukemia inhibitory factor (LIF). LIF activates the JAK‐STAT3 pathway through the class I cytokine receptor gp130, which forms a trimeric complex with LIF and the class I cytokine receptor LIF receptor β. STAT3 has been shown to play a crucial role in self‐renewal in mouse ES cells probably by induction of c‐myc expression. Thus, ablation of STAT3 activation leads to differentiation. However, important connections between STAT3 and other signalling pathways have been documented. In addition, gp130 activation leads to both PI3K and Src activation. The canonical Wnt pathway is sufficient to maintain self‐renewal of both human ES cells and mouse ES cells. It seems quite possible that the main pathway maintaining self‐renewal in ES cells is the Wnt pathway, while the LIF‐JAK‐STAT3 pathway is present in mouse cells as an adaptation for sustaining self‐renewal during embryonic diapause, a condition of delayed implantation in mammals. In keeping with this scenario, the Wnt pathway has been shown to elevate the level of c‐myc. Thus, the two pathways seem to converge on c‐myc as a common target to promote self‐renewal. Whereas LIF does not seem to stimulate self‐renewal in human embryonic stem cells it cannot be excluded that other cytokines are involved. The pleiotropic actions of the increasing number of cytokines and receptors signalling via JAKs, STATs and SOCS exhibit considerable redundancy, compensation and plasticity in stem cells in accordance with the view that stem cells are governed by quantitative variations in strength and duration of signalling events known from other cell types rather than qualitatively different stem cell‐specific factors.

Many Putative Endocrine Disruptors Inhibit Prostaglandin Synthesis

Background Prostaglandins (PGs) play key roles in development and maintenance of homeostasis of the adult body. Despite these important roles, it remains unclear whether the PG pathway is a target for endocrine disruption. However, several known endocrine-disrupting compounds (EDCs) share a high degree of structural similarity with mild analgesics. Objectives and Methods Using cell-based transfection and transduction experiments, mass spectrometry, and organotypic assays together with molecular modeling, we investigated whether inhibition of the PG pathway by known EDCs could be a novel point of endocrine disruption. Results We found that many known EDCs inhibit the PG pathway in a mouse Sertoli cell line and in human primary mast cells. The EDCs also reduced PG synthesis in ex vivo rat testis, and this reduction was correlated with a reduced testosterone production. The inhibition of PG synthesis occurred without involvement of canonical PG receptors or the peroxisome proliferator–activated receptors (PPARs), which have previously been described as targets of EDCs. Instead, our results suggest that the compounds may bind directly into the active site of the cyclooxygenase (COX) enzymes, thereby obstructing the conversion of arachidonic acid to PG precursors without interfering with the expression of the COX enzymes. A common feature of the PG inhibitory EDCs is the presence of aromatic groups that may stabilize binding in the hydrophobic active site of the COX enzymes. Conclusion Our findings suggest a hitherto unknown mode of action by EDCs through inhibition of the PG pathway and suggest new avenues to investigate effects of EDCs on reproductive and immunological disorders that have become increasingly common in recent decades.

Paracetamol (acetaminophen), aspirin (acetylsalicylic acid) and indomethacin are anti‐androgenic in the rat foetal testis

More than half the pregnant women in the Western world report taking mild analgesics. These pharmaceutical compounds have been associated with congenital cryptorchidism in humans, the best-known risk factor for low semen quality and testicular germ cell cancer. Furthermore, some of these mild analgesics exert potent anti-androgenic effects in the male rat and several endocrine-disrupting compounds, known to alter masculinization, have also been shown to be potent inhibitors of prostaglandin (PG) synthesis similar to mild analgesics. Using a 3-day ex vivo organotypic model system based on gestational day 14.5 rat testes, we herein show that testosterone production was inhibited by paracetamol, at doses of 0.1 μm to 100 μm. Similar results were obtained for aspirin (1-100 μm) and indomethacin (10 μm). The production of the other Leydig cell hormone, Insl3, was not disrupted by exposure to paracetamol. Investigations of the gross anatomy of the testis as well as Leydig cells number and rate of gonocyte apoptosis after the 3 days of ex vivo differentiation showed no significant effect of the analgesics tested compared with controls. These data indicate therefore that mild analgesics specifically inhibit testosterone production in rat foetal testes in vitro and that these compounds had no effect on gonocyte survival. Parallel determinations of prostaglandin D2 (PGD2) production indicated that the effects of paracetamol and aspirin on PGD2 and testosterone were not connected, whereas the effects of indomethacin were correlated. We conclude that mild analgesics exert direct and specific anti-androgenic effects in rat foetal testis in our experimental setup and that the mechanism of action is probably uncoupled from the inhibition of PG synthesis.

Testicular dysgenesis syndrome and the origin of carcinoma in situ testis

Recent increases in male reproductive disorders have been linked to exposure to environmental factors leading to the testicular dysgenesis syndrome (TDS). Testicular cancer is the most severe condition in TDS and studies have shown a clear correlation between risk of testicular cancer and other components of TDS and that the geographical location of the mother during pregnancy can be a risk factor. This suggests that the dysgenesis has its origin in utero and that TDS is initiated by environmental factors, including possibly hormone-disrupting compounds that act on the mother and the developing foetus, but the genetic background may also play a role. The morphological similarity of carcinoma in situ (CIS) cells (the precursor of the majority of invasive testicular cancers) with primordial germ cells and gonocytes, and overlap in expression of protein markers suggests an origin of CIS from primordial germ cells or gonocytes. CIS cells and germ cell-derived cancers of the human type have so far not been described in any animal model of TDS, which could be caused by species differences in the development of the male gonad. Regardless of this, it is plausible that the dysgenesis, and hence the development of CIS cells, is a result of disturbed signalling between nurse cells and germ cells that allow embryonic germ cells to survive in the pre-pubertal and adult testis. The post-pubertal proliferation of CIS cells combined with aberrant signalling then leads to an accumulation of genetic changes in the CIS cells, which eventually results in the development of invasive testicular cancer in the adult.

Analysis of SOX2 expression in developing human testis and germ cell neoplasia.

The transcriptional regulators of pluripotency, POU5F1 (OCT4), NANOG and SOX2, are highly expressed in embryonal carcinoma (EC). In contrast to OCT4 and NANOG, SOX2 has not been demonstrated in the early human germ cell lineage or carcinoma in situ (CIS), the precursor for testicular germ cell tumours (TGCTs). Here, we have analysed SOX2 expression in CIS and overt TGCTs, as well as normal second and third trimester fetal, prepubertal and adult testes by in situ hybridisation and immunohistochemistry using three different antibodies. In contrast to earlier studies, we detected SOX2 mRNA in most CIS cells. We also detected speckled nuclear SOX2 immunoreactivity in CIS cells with one primary antibody, which was not apparent with other primary antibodies. The results demonstrate SOX2 gene expression in CIS for the first time and raise the possibility of post-transcriptional regulation, most likely sumoylation as a mechanism for limiting SOX2 action in these cells.