High incidence of imperforate vagina in ADGRA3-deficient mice.

BACKGROUND: Ten percent of the female population suffers from congenital abnormalities of the vagina, uterus, or oviducts, with severe consequences for reproductive and psychological health. Yet, the underlying causes of most of these malformations remain largely unknown. ADGRA3 (GPR125) is involved in WNT signaling and planar cell polarity, mechanisms vital to female reproductive tract development. Although ADGRA3 is a well-established spermatogonial stem cell marker, its role within the female urogenital system remains unclear. RESULTS: In this study, we found Adgra3 to be expressed throughout the murine female urogenital system, with higher expression pre-puberty than after sexual maturation. We generated a global Adgra3-/- mouse line and observed imperforate vagina in 44% of Adgra3-/- females, resulting in distension of the reproductive tract and infertility. Ovarian morphology, plasma estradiol, ovarian Cyp19a1, and vaginal estrogen receptor α (Esr1) expression were unaffected. However, compared to controls, a significantly lower bone mineral density was found in Adgra3-/- mice. Whereas vaginal opening in mice is an estrogen-dependent process, 17ß-estradiol treatment failed to induce vaginal canalization in Adgra3-/- mice. Furthermore, a marked reduction in vaginal and ovarian progesterone receptor expression was observed concomitant with an upregulation of apoptotic regulators Bcl2, Bid, and Bmf in adult Adgra3-/- females with a closed vagina. CONCLUSIONS: Our collective results shed new insights into the complex mechanisms by which the adhesion receptor ADGRA3 regulates distal vaginal tissue remodeling during vaginal canalization via altered sex hormone responsiveness and balance in apoptotic regulators. This highlights the potential of ADGRA3 as a target in diagnostic screening and/or therapy for obstructive vaginal malformations in humans.

Calcium transport in male reproduction is possibly influenced by vitamin D and CaSR.

Vitamin D is important for gonadal function in rodents, and improvement of vitamin D status in men with low sperm counts increases live birth rate. Vitamin D is a regulator of transcellular calcium transport in the intestine and kidney and may influence the dramatic changes in the luminal calcium concentration in epididymis. Here, we show spatial expression in the male reproductive tract of vitamin D receptor (VDR)-regulated factors involved in calcium transport: transient receptor potential vanilloid 5/6 , sodium/calcium exchanger 1, plasma membrane calcium ATPase 1, calbindin D9k, calcium-sensing receptor (CaSR), and parathyroid hormone-related peptide (PTHrP) in mouse and human testis and epididymis. Testicular Casr expression was lower in Vdr ablated mice compared with controls. Moreover, expression levels of Casr and Pthrp were strongly correlated in both testis and epididymis and Pthrp was suppressed by 1,25(OH)2D3 in a spermatogonial cell line. The expression of CaSR in epididymis may be of greater importance than in the gonad in mice as germ cell-specific Casr deficient mice had no major reproductive phenotype, and coincubation with a CaSR-agonist had no effect on human sperm-oocyte binding. In humans, seminal calcium concentration between 5 and 10 mM was associated with a higher fraction of motile and morphologically normal sperm cells, and the seminal calcium concentration was not associated with serum calcium levels. In conclusion, VDR regulates CaSR and PTHrP, and both factors may be involved in the regulation of calcium transport in the male reproductive tract with possible implications for sperm function and storage.

RANKL regulates male reproductive function.

Infertile men have few treatment options. Here, we demonstrate that the transmembrane receptor activator of NF-kB ligand (RANKL) signaling system is active in mouse and human testis. RANKL is highly expressed in Sertoli cells and signals through RANK, expressed in most germ cells, whereas the RANKL-inhibitor osteoprotegerin (OPG) is expressed in germ and peritubular cells. OPG treatment increases wild-type mouse sperm counts, and mice with global or Sertoli-specific genetic suppression of Rankl have increased male fertility and sperm counts. Moreover, RANKL levels in seminal fluid are high and distinguishes normal from infertile men with higher specificity than total sperm count. In infertile men, one dose of Denosumab decreases RANKL seminal fluid concentration and increases serum Inhibin-B and anti-Müllerian-hormone levels, but semen quality only in a subgroup. This translational study suggests that RANKL is a regulator of male reproductive function, however, predictive biomarkers for treatment-outcome requires further investigation in placebo-controlled studies.

The Calcium-Sensing Receptor Is Essential for Calcium and Bicarbonate Sensitivity in Human Spermatozoa.

CONTEXT: The calcium-sensing receptor (CaSR) is essential to maintain a stable calcium concentration in serum. Spermatozoa are exposed to immense changes in concentrations of CaSR ligands such as calcium, magnesium, and spermine during epididymal maturation, in the ejaculate, and in the female reproductive environment. However, the role of CaSR in human spermatozoa is unknown. OBJECTIVE: This work aimed to investigate the role of CaSR in human spermatozoa. METHODS: We identified CaSR in human spermatozoa and characterized the response to CaSR agonists on intracellular calcium, acrosome reaction, and 3',5'-cyclic adenosine 5'-monophosphate (cAMP) in spermatozoa from men with either loss-of-function or gain-of-function mutations in CASR and healthy donors. RESULTS: CaSR is expressed in human spermatozoa and is essential for sensing extracellular free ionized calcium (Ca2+) and Mg2+. Activators of CaSR augmented the effect of sperm-activating signals such as the response to HCO3- and the acrosome reaction, whereas spermatozoa from men with a loss-of-function mutation in CASR had a diminished response to HCO3-, lower progesterone-mediated calcium influx, and were less likely to undergo the acrosome reaction in response to progesterone or Ca2+. CaSR activation increased cAMP through soluble adenylyl cyclase (sAC) activity and increased calcium influx through CatSper. Moreover, external Ca2+ or Mg2+ was indispensable for HCO3- activation of sAC. Two male patients with a CASR loss-of-function mutation in exon 3 presented with normal sperm counts and motility, whereas a patient with a loss-of-function mutation in exon 7 had low sperm count, motility, and morphology. CONCLUSION: CaSR is important for the sensing of Ca2+, Mg2+, and HCO3- in spermatozoa, and loss-of-function may impair male sperm function.

Novel functions of the luteinizing hormone/chorionic gonadotropin receptor in prostate cancer cells and patients.

Prostate cancer (PCa) cells become castrate-resistant after initial tumor regression following castration-based lowering of testosterone (T). De-novo intra-tumoral steroid synthesis is a suggested biological mechanism of castration resistant PCa, but the regulators are unknown. Testicular T production is controlled by the luteinizing hormone/choriogonadotropin receptor (LHCGR). To elucidate the influence of LHCGR on PCa development the presence and effects of LHCGR in PCa and whether LHCGR in serum holds prognostic information in PCa patients is investigated. LHCGR expression was investigated by RT-PCR, WB, IHC, qPCR in PCa cell lines and prostatic tissue. Steroid production was measured in media from cell lines with LC-MS/MS and expression of steroidogenic enzymes with qPCR. Serum LHCGR (sLHCGR) was measured with ELISA in PCa patients (N = 157). Presence of LHCGR was established in prostatic tissue and PCa cell lines. Cell proliferation increased by 1.29-fold in LNCaP (P = 0.007) and 1.33-fold in PC-3 cells (P = 0.0007), when stimulated by luteinizing hormone. Choriogonadotropin stimulation decreased proliferation 0.93-fold in DU145 cells (P = 0.05), but none of the treatments altered steroid metabolite secretion. Low sLHCGR concentration was associated with a higher risk of biochemical failure after radical prostatectomy (HR = 3.05, P = 0.06) and castration resistance (HR = 6.92, P = 0.004) compared to high sLHCGR concentration. LHCGR is expressed in PCa and may exert a growth regulatory role in PCa derived cell lines. A potential prognostic role of sLHCGR for determining recurrence risk in PCa patients is found in this pilot study but needs verification in larger cohorts.

Luteinizing Hormone Receptor Is Expressed in Testicular Germ Cell Tumors: Possible Implications for Tumor Growth and Prognosis.

Luteinizing hormone/choriogonadotropin receptor (LHCGR) regulates gonadal testosterone production and recent studies have suggested a growth-regulatory role in somatic cancers. Here, we established that LHCGR is expressed in a fraction of seminoma cells and germ cell neoplasia in situ (GCNIS), and the seminoma-derived cell line TCam2 released LHCGR into the medium. LH treatment induced proliferation of TCam2 cells in vitro, while hCG treatment induced a non-significant 51% increase in volume of tumors formed in a TCam2 xenograft model. A specific ELISA was used to detect a soluble LHCGR in serum. Serum concentrations of soluble LHCGR could not distinguish 4 patients with GCNIS and 216 patients with testicular germ cell tumors (TGCTs) from 297 infertile or 148 healthy young men. Instead, serum LHCGR levels were significantly higher in 112 patients with a seminoma > 5 cm or elevated serum lactate dehydrogenase (LDH) compared with men harboring smaller seminomas < 2 cm or normal LDH levels. Serum LHCGR levels in TGCT patients could not predict relapse irrespective whether determined pre- or post-orchiectomy. Combined, these novel findings suggest that LHCGR may be directly involved in the progression and growth of seminomas, and our retrospective pilot study suggests that serum LHCGR may have some prognostic value in men with seminoma.

Vitamin D and sex steroid production in men with normal or impaired Leydig cell function.

Production of testosterone is under tight control by human chorion gonadotropin (hCG) during fetal life and luteinizing hormone (LH) in adulthood. Several animal and human studies have linked vitamin D status with sex steroid production although it is not clear whether there exist a direct or indirect involvement in androgen production. Few studies have investigated this crosslink in young healthy men and putative direct or synergistic effect of activated vitamin D (1,25(OH)2D3) and LH/hCG on sex steroid production in vitro. Here, we present cross-sectional data from 300 young men and 41 hCG-stimulated men with impaired Leydig cell function combined with data from an ex vivo culture of human testicular tissue exposed to 1,25(OH)2D3 alone or in combination with hCG. Serum 25-OHD was positively associated with SHBG (ß:0.002; p = 0.023) and testosterone/estradiol-ratio (ß:0.001; p = 0.039), and inversely associated with free testosterone (%) (free testosterone/total testosterone) (ß:-0.002; p = 0.016) in young men. Vitamin D deficient men had higher total and free estradiol concentrations than men with higher vitamin D status (19% and 18%, respectively; p < 0.01). Interestingly, men with impaired Leydig cell function and vitamin D deficiency had a significantly lower hCG-mediated increase in total and free testosterone compared with vitamin D sufficient men (p < 0.05). Accordingly, testicular tissue exposed to 100 nM 1,25(OH)2D3 had a 15% higher testosterone release into the media compared with vehicle treated specimens (p = 0.030). In conclusion, vitamin D deficiency is associated with lower testosterone/estradiol ratio in young men and lower Leydig cell sensitivity after hCG-stimulation in men with impaired gonadal function. The significant effect of 1,25(OH)2D3 on testosterone production in a human testis model supports that the stimulatory effect at least in part may be direct. Larger placebo-controlled studies are needed to determine whether vitamin D supplementation can influence testosterone production.

Expression pattern of clinically relevant markers in paediatric germ cell- and sex-cord stromal tumours is similar to adult testicular tumours.

Paediatric germ cell tumours (GCTs) are rare and account for less than 3 % of childhood cancers. Like adult GCTs, they probably originate from primordial germ cells, but the pattern of histopathological types is different, and they occur predominantly in extragonadal sites along the body midline. Because they are rare, histology of paediatric GCTs is poorly documented, and it remains unclear to what extent they differ from adult GCTs. We have analysed 35 paediatric germ cell tumours and 5 gonadal sex-cord stromal tumours from prepubertal patients aged 0-15 years, to gain further knowledge, elaborate on clinical-pathological associations and better understand their developmental divergence. The tumours were screened for expression of stemness-related factors (OCT4, AP-2γ, SOX2), classical yolk sac tumours (YSTs; AFP, SALL4), GCTs (HCG, PLAP, PDPN/D2-40), as well as markers for sex-cord stromal tumour (PDPN, GATA4). All YSTs expressed AFP and SALL4, with GATA4 present in 13/14. The majority of teratomas expressed SOX2 and PDPN, whereas SALL4 was found in 8/13 immature teratomas. Adult seminoma markers AP-2γ, OCT4, SALL4 and PDPN were all expressed in dysgerminoma. We further report a previously unrecognised pathogenetic relationship between AFP and SALL4 in YST in that different populations of YST cells express either SALL4 or AFP, which suggests variable differentiation status. We also show that AP-2γ is expressed in the granulosa layer of ovarian follicles and weakly expressed in immature but not in mature granulosa cell tumours. Our findings indicate that the expression pattern of these antigens is similar between paediatric and adult GCTs, even though they develop along different developmental trajectories.

Transcriptome profiling of mice testes following low dose irradiation.

BACKGROUND: Radiotherapy is used routinely to treat testicular cancer. Testicular cells vary in radio-sensitivity and the aim of this study was to investigate cellular and molecular changes caused by low dose irradiation of mice testis and to identify transcripts from different cell types in the adult testis. METHODS: Transcriptome profiling was performed on total RNA from testes sampled at various time points (n = 17) after 1 Gy of irradiation. Transcripts displaying large overall expression changes during the time series, but small expression changes between neighbouring time points were selected for further analysis. These transcripts were separated into clusters and their cellular origin was determined. Immunohistochemistry and in silico quantification was further used to study cellular changes post-irradiation (pi). RESULTS: We identified a subset of transcripts (n = 988) where changes in expression pi can be explained by changes in cellularity. We separated the transcripts into five unique clusters that we associated with spermatogonia, spermatocytes, early spermatids, late spermatids and somatic cells, respectively. Transcripts in the somatic cell cluster showed large changes in expression pi, mainly caused by changes in cellularity. Further investigations revealed that the low dose irradiation seemed to cause Leydig cell hyperplasia, which contributed to the detected expression changes in the somatic cell cluster. CONCLUSIONS: The five clusters represent gene expression in distinct cell types of the adult testis. We observed large expression changes in the somatic cell profile, which mainly could be attributed to changes in cellularity, but hyperplasia of Leydig cells may also play a role. We speculate that the possible hyperplasia may be caused by lower testosterone production and inadequate inhibin signalling due to missing germ cells.

Influence of vitamin D on cisplatin sensitivity in testicular germ cell cancer-derived cell lines and in a NTera2 xenograft model.

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has anti-proliferative, pro-apoptotic, and pro-differentiating effects in somatic cancer cells in vitro and in vivo. 1,25(OH)2D3 also augments the anti-tumor effects of several chemotherapeutic agents, including cisplatin, which may have clinical relevance. Given the pro-differentiation effect of vitamin D recently demonstrated in testicular germ cell tumors (TGCTs), we hypothesized that 1,25(OH)2D3 could be a beneficial adjunctive to existing chemotherapy regime used to treat these tumors. In this study, cell survival effects of 1,25(OH)2D3, another pro-differentiation compound, retinoic acid and cisplatin were investigated in TGCT-derived cell lines in vitro. 1,25(OH)2D3 augmented the effect of cisplatin in an embryonal carcinoma-derived cell line (NTera2), possibly through downregulation of pluripotency genes and simultaneous upregulation of the cell cycle regulators p21, p27, p53, p73 and FOXO1, while no significant effects were found in TCam-2 and 2102Ep cell lines (derived from seminoma and embryonal carcinoma, respectively). Anti-tumor effects of cholecalciferol, 1,25(OH)2D3, and cisplatin were subsequently tested in vivo, in a NTera2 xenograft tumor model in nude mice. In xenograft tumors, co-treatment with 1,25(OH)2D3 and cisplatin resulted in downregulation of OCT4 and simultaneous upregulation of p21 and p73, but did not reduce tumor growth significantly more than cisplatin alone. Also, cholecalciferol supplemented diet (1100IU daily) after tumor formation did not increase cisplatin sensitivity in vivo. In conclusion, addition of 1,25(OH)2D3 augmented the antitumor effect of cisplatin monotherapy in vitro, but not in this in vivo testicular germ cell cancer model. Future studies are needed to investigate potential beneficial effects of vitamin D with lower cisplatin doses, and to determine whether 1,25(OH)2D3 may increase cisplatin sensitivity in chemotherapy-resistant TGCTs. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Vitamin D metabolism and effects on pluripotency genes and cell differentiation in testicular germ cell tumors in vitro and in vivo.

Testicular germ cell tumors (TGCTs) are classified as either seminomas or nonseminomas. Both tumors originate from carcinoma in situ (CIS) cells, which are derived from transformed fetal gonocytes. CIS, seminoma, and the undifferentiated embryonal carcinoma (EC) retain an embryonic phenotype and express pluripotency factors (NANOG/OCT4). Vitamin D (VD) is metabolized in the testes, and here, we examined VD metabolism in TGCT differentiation and pluripotency regulation. We established that the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human fetal germ cells, CIS, and invasive TGCTs. VD metabolism diminished markedly during the malignant transformation from CIS to EC but was reestablished in differentiated components of nonseminomas, distinguished by coexpression of mesodermal markers and loss of OCT4. Subsequent in vitro studies confirmed that 1,25(OH)(2)D(3) (active VD) downregulated NANOG and OCT4 through genomic VDR activation in EC-derived NTera2 cells and, to a lesser extent, in seminoma-derived TCam-2 cells, and up-regulated brachyury, SNAI1, osteocalcin, osteopontin, and fibroblast growth factor 23. To test for a possible therapeutic effect in vivo, NTera2 cells were xenografted into nude mice and treated with 1,25(OH)(2)D(3), which induced down-regulation of pluripotency factors but caused no significant reduction of tumor growth. During NTera2 tumor formation, down-regulation of VDR was observed, resulting in limited responsiveness to cholecalciferol and 1,25(OH)(2)D(3) treatment in vivo. These novel findings show that VD metabolism is involved in the mesodermal transition during differentiation of cancer cells with embryonic stem cell characteristics, which points to a function for VD during early embryonic development and possibly in the pathogenesis of TGCTs.

Optimizing staining protocols for laser microdissection of specific cell types from the testis including carcinoma in situ.

Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser microdissection technology allows for enrichment of specific cell types. However, when the cells are not morphologically distinguishable, it is necessary to use a specific staining method for the target cells. In this study we have tested different fixatives, storage conditions for frozen sections and staining protocols, and present two staining protocols for frozen sections, one for fast and specific staining of fetal germ cells, testicular carcinoma in situ cells, and other cells with embryonic stem cell-like properties that express the alkaline phosphatase, and one for specific staining of lipid droplet-containing cells, which is useful for isolation of the androgen-producing Leydig cells. Both protocols retain a morphology that is compatible with laser microdissection and yield RNA of a quality suitable for PCR and microarray analysis.