Anne Jørgensen

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

BACKGROUNDnThe 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.nnnMETHODSnTissue 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.nnnRESULTSnVDR 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.nnnCONCLUSIONnOn 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.

Expression profiles for six zebrafish genes during gonadal sex differentiation

BackgroundThe mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation.ResultsIn the present study, we have used quantitative real-time PCR to investigate the expression of ar, sox9a, dmrt1, fig alpha, cyp19a1a and cyp19a1b during the expected sex determination and gonadal sex differentiation period. The expression of the genes expected to be high in males (ar, sox9a and dmrt1a) and high in females (fig alpha and cyp19a1a) was segregated in two groups with more than 10 times difference in expression levels. All of the investigated genes showed peaks in expression levels during the time of sex determination and gonadal sex differentiation. Expression of all genes was investigated on cDNA from the same fish allowing comparison of the high and low expressers of genes that are expected to be highest expressed in either males or females. There were 78% high or low expressers of all three male genes (ar, sox9a and dmrt1) in the investigated period and 81% were high or low expressers of both female genes (fig alpha and cyp19a1a). When comparing all five genes with expected sex related expression 56% show expression expected for either male or female. Furthermore, the expression of all genes was investigated in different tissue of adult male and female zebrafish.ConclusionIn zebrafish, the first significant peak in gene expression during the investigated period (2–40 dph) was dmrt1 at 10 dph which indicates involvement of this gene in the early gonadal sex differentiation of males.

Characterization of the testicular, epididymal and endocrine phenotypes in the Leuven Vdr-deficient mouse model: targeting estrogen signalling.

Vitamin D is a key factor for calcium and bone homeostasis, but signalling through the vitamin D receptor (VDR) seems also to be important for testicular function. To test the functional role of vitamin D signalling we examined the male reproductive system of the Leuven Vdr-ablated (Vdr(-/-)) mice, previously established as a model for hereditary vitamin D-resistant rickets. We investigated reproductive hormones, changes in gene expression and histological phenotype of eleven Vdr(-/-), eight Vdr(+/-) and nine Vdr(+/+) mice. Testicular and epididymal histology were grossly normal in Vdr(-/-) mice. Accordingly, no differences were found in serum concentrations of testosterone, estradiol, LH, and FSH or testicular expression of Cyp19a1, Ersα, Cyp17a1, Star, Insl3, Inhbb, and Amh. However, a significantly lower ERβ expression was found in testis of Vdr(+/-) and Vdr(-/-) mice, conversely epididymal expressions of ERα and the estrogen-target gene Aqp9 were higher. In conclusion, vitamin D seems dispensable for murine spermatogenesis and sex hormone production, but aberrant estrogen-signalling may elicit some of the VDR-mediated effects on male reproduction.

Anti-mullerian hormone and its clinical use in pediatrics with special emphasis on disorders of sex development.

Using measurements of circulating anti-Müllerian hormone (AMH) in diagnosing and managing reproductive disorders in pediatric patients requires thorough knowledge on normative values according to age and gender. We provide age- and sex-specific reference ranges for the Immunotech assay and conversion factors for the DSL and Generation II assays. With this tool in hand, the pediatrician can use serum concentrations of AMH when determining the presence of testicular tissue in patients with bilaterally absent testes or more severe Disorders of Sex Development (DSD). Furthermore, AMH can be used as a marker of premature ovarian insufficiency (POI) in both Turner Syndrome patients and in girls with cancer after treatment with alkylating gonadotoxic agents. Lastly, its usefulness has been proposed in the diagnosis of polycystic ovarian syndrome (PCOS) and ovarian granulosa cell tumors and in the evaluation of patients with hypogonadotropic hypogonadism.

TBT pollution and effects in molluscs at US Virgin Islands, Caribbean Sea

An almost ubiquitous occurrence of imposex and butyltins in the molluscs from US Virgin Islands gives evidence to a widespread contamination with the antifouling agent tributyltin (TBT), which most likely is related to a relatively intense ship traffic. Three different muricid neogastropod species Thais deltoidea, Thais rustica and Purpura patula all seem to have potential as suitable and sensitive bioindicators for assessing levels and effects of TBT pollution in coastal areas including coral reefs in the Caribbean Sea. However, considerable interspecies differences in especially accumulation potential of butyltins were seen in this study. Furthermore, a high accumulation potential of TBT in the edible gastropod West Indian topshell (Cittarium pica) was found, despite that no signs of imposex were observed in this archaeogastropod species.

Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche.

Background:Testicular germ cell tumours of young adults, seminoma or non-seminomas, are preceded by a pre-invasive precursor, carcinoma in situ (CIS), understood to arise through differentiation arrest of embryonic germ cells. Knowledge about the malignant transformation of germ cells is currently limited by the lack of experimental models. The aim of this study was to establish an experimental tissue culture model to maintain normal and malignant germ cells within their niche and allow investigation of treatment effects.Methods:Human testis and testis cancer specimens from orchidectomies were cultured in ‘hanging drops’ and effects of activin A and follistatin treatment were investigated in seminoma cultures.Results:Testis fragments with normal spermatogenesis or CIS cells were cultured for 14 days with sustained proliferation of germ cells and CIS cells and without increased apoptosis. Seminoma cultures survived 7 days, with proliferating cells detectable during the first 5 days. Activin A treatment significantly reduced KIT transcript and protein levels in seminoma cultures, thereby demonstrating a specific treatment response.Conclusions:Hanging drop cultures of human testis and testis cancer samples can be employed to delineate mechanisms governing growth of normal, CIS and tumorigenic germ cells retained within their niche.

Identification of a Novel Androgen Receptor Mutation in a Family With Multiple Components Compatible With the Testicular Dysgenesis Syndrome

CONTEXTnAndrogen signaling via the androgen receptor (AR) is essential for normal testis development and male reproductive functions. We describe a rare family with 3 males affected by a mild disorder of sex determination compatible with testicular dysgenesis syndrome (TDS), including subfertility, cryptorchidism, hypospadias, and testicular cancer, caused by a novel AR mutation.nnnOBJECTIVEnThe aim of this study was to describe the phenotype of the affected males, characterize functionally the novel AR mutation, and discuss the significance of partial androgen insufficiency in the pathogenesis of TDS.nnnPARTICIPANTSnThe proband, his first cousin, and a nephew underwent a detailed clinical investigation including genetic tests, whereas four female members of the family were tested for the specific AR mutation.nnnRESULTSnA novel AR mutation, c.2214T>G;p.Ile738Met, was identified in the affected family members. Functional analysis of the mutation in a gene-reporter assay showed a 50% reduction in AR-induced transcriptional activity. The affected males had elevated LH and T in accordance with decreased AR signaling. The histology and immunohistochemical profile of the testis tissue from the 2 patients with testicular cancer showed features consistent with insufficient testis development and TDS.nnnCONCLUSIONnThe presence of all hallmarks of TDS, including germ cell cancer, in a family with a novel AR mutation causing a partial decrease in AR function is in line with the concept that reduced androgen signaling may contribute to the development of TDS. It also seems consistent with the hypothesis that environmental factors interfering with this pathway can play a role in the pathogenesis of TDS.

Laser capture microdissection of gonads from juvenile zebrafish

BackgroundInvestigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation.MethodsThe laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads.ResultsThe juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling.ConclusionThe study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species.

Expression of prostaglandin synthases (pgds and pges) during zebrafish gonadal differentiation.

The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E2 synthase (pges) and especially the lipocalin-type prostaglandin D2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found in other species. In mice and chicken, the lipocalin-type Pgds is specifically expressed in pre-Sertoli cells just after Sry and Sox9 and is involved in masculinisation of the developing testis. Furthermore, Pges are implicated in female reproduction including follicular development and ovulation. In this study, a sexually dimorphic expression of pgds was found in gonads of adult zebrafish with expression in testis but not in ovaries. To determine whether the sex-specific expression pattern of pgds was present in gonads of juvenile zebrafish and therefore could be an early marker of sex in zebrafish, we microdissected gonads from four randomly selected individual zebrafish for every second day in the period 2-20 days post hatch (dph) and 0-1 dph. The temporal expression of pgds and pges was investigated in the microdissected gonads, however, no differential expression that could indicate sex-specific difference between individual juvenile zebrafish was observed.

Interaction between paraoxonase 1 polymorphism and prenatal pesticide exposure on metabolic markers in children using a multiplex approach

Prenatal environmental exposures may influence the risk of cardio-metabolic diseases later in life. This study used a multiplex approach to investigate non-fasting serum levels of metabolic markers in a cohort of school-aged children for whom associations between prenatal pesticide exposure and body fat content and blood pressure were previously found to be dependent on paraoxonase1 (PON1) Q192R genotype. In children with the PON1 192 R-allele, leptin, glucagon, and plasminogen activator inhibitor-1 (PAI-1) were positively associated with prenatal pesticide exposure. For PON1 192 QQ-homozygote children none of the biomarkers were significantly affected by prenatal pesticide exposure. In children with the R-allele, leptin was associated with both body fat measures and prenatal pesticide exposure and seems to mediate body fat accumulation in exposed children. These findings support our previous results of an adverse cardio-metabolic risk profile associated with prenatal pesticide exposure in children with the PON1 192 R-allele.