Testicular immune cell populations and macrophage polarisation in adult male mice and the influence of altered activin A levels.

Detailed morphological characterization of testicular leukocytes in the adult CX3CR1 gfp/+ transgenic mouse identified two distinct CX3CR1 + mononuclear phagocyte (macrophage and dendritic cell) populations: stellate/dendriform cells opposed to the seminiferous tubules (peritubular), and polygonal cells associated with Leydig cells (interstitial). Using confocal microscopy combined with stereological enumeration of CX3CR1gfp/+ cells established that there were twice as many interstitial cells (68%) as peritubular cells (32%). Flow cytometric analyses of interstitial cells from mechanically-dissociated testes identified multiple mononuclear phagocyte subsets based on surface marker expression (CX3CR1, F4/80, CD11c). These cells comprised 80% of total intratesticular leukocytes, as identified by CD45 expression. The remaining leukocytes were CD3+ (T lymphocytes) and NK1.1+ (natural killer cells). Functional phenotype assessment using CD206 (an anti-inflammatory/M2 marker) and MHC class II (an activation marker) identified a potentially tolerogenic CD206+MHCII+ sub-population (12% of total CD45+ cells). Rare testicular subsets of CX3CR1 +CD11c+F4/80+ (4.3%) mononuclear phagocytes and CD3+NK1.1+ (3.1%) lymphocytes were also identified for the first time. In order to examine the potential for the immunoregulatory cytokine, activin A to modulate testicular immune cell populations, testes from adult mice with reduced activin A (Inhba+/-) or elevated activin A (Inha+/-) were assessed using flow cytometry. Although the proportion of F4/80+CD11b+ leukocytes (macrophages) was not affected, the frequency of CD206+MHCII+cells was significantly lower and CD206+MHCII- correspondingly higher in Inha+/- testes. This shift in expression of MHCII in CD206+ macrophages indicates that changes in circulating and/or local activin A influence resident macrophage activation and phenotype and, therefore, the immunological environment of the testis.

Immunolocalization of DMRTB1 in human testis with normal and impaired spermatogenesis.

BACKGROUND: The transcription factor DMRTB1 plays a pivotal role in coordinating the transition between mitosis and meiosis in murine germ cells. No reliable data are available for human testis. OBJECTIVES: The present study aims to examine the testicular expression pattern of DMRTB1 in men showing normal and impaired spermatogenesis. MATERIALS AND METHODS: Immunohistochemistry was performed using 54 human testicular biopsy specimens and a commercial rabbit polyclonal anti-DMRTB1 primary antibody. RT-PCR complemented immunohistochemistry. To further characterize immunopositive cells and possible co-localization, the proliferation marker Ki-67, the tumor marker PLAP, and an anti-DMRT1 antibody were used. RESULTS: In men with normal spermatogenesis, a strong immunoreactivity was detectable in a subset of spermatogonia (38.34 ± 2.14%). Some spermatocytes showed a weak immunostaining. Adjacent Sertoli cells were immunonegative. Compared with a hematoxylin and eosin overview staining, these immunopositive cells were almost exclusively identified as Apale and B spermatogonia and primary spermatocytes in (pre-)leptotene, zygotene, and pachytene stages. In patients with spermatogenic arrest at spermatogonial level, an altered staining pattern was found. No immunoreactivity was detected in Sertoli cells in Sertoli cell-only syndrome. In germ cell neoplasia in situ (GCNIS) tubules, except for a few (0.4 ± 0.03%), pre-invasive tumor cells were immunonegative. Seminoma cells showed no immunostaining. DISCUSSION: According to previous findings in mice, it seems reasonable that DMRTB1 is expressed in these normal germ cell populations. Moreover, altered staining pattern in spermatogenic arrest at spermatogonial stage suggests a correlation with mitosis and transformation into B spermatogonia. The absence of DMRTB1 in GCNIS cells and tumor cells might be associated with uncontrolled neoplastic cell proliferation and progression into invasive germ cell tumors. Further research is required to elucidate, for example, the role of DMRTB1 in the malignant transformation of human germ cells. CONCLUSION: Our data indicate a relevant role for DMRTB1 regarding the entry of spermatogonia into meiosis in men.

Transporter for sulfated steroid hormones in the testis - expression pattern, biological significance and implications for fertility in men and rodents.

In various tissues, steroid hormones may be sulfated, glucuronidated or otherwise modified. For a long time, these hydrophilic molecules have been considered to be merely inactive metabolites for excretion via bile or urine. Nevertheless, different organs such as the placenta and breast tissue produce large amounts of sulfated steroids. After the discovery of the enzyme steroid sulfatase, which is able to re-activate sulfated steroids, these precursor molecules entered the focus of interest again as a local supply for steroid hormone synthesis with a prolonged half-life compared to their unconjugated counterparts. The first descriptions of this so-called sulfatase pathway in the placenta and breast tissue (with special regards to hormone-dependent breast cancer) were quickly followed by studies of steroid sulfate production and function in the testis. These hydrophilic molecules may not permeate the cell membrane by diffusion in the way that unbound steroids can, but need to be transported through the plasma membrane by transport systems. In the testis, a functional sulfatase pathway requires the expression of specific uptake carrier and efflux transporters in testicular cells, i.e. Sertoli, Leydig and germ cells. Main focus has to be placed on Sertoli cells, as these cells build up the blood-testis barrier. In this review, an overview of carrier expression pattern in the human as well as rodent testis is provided with special interest towards implications on fertility.

Current insights into the sulfatase pathway in human testis and cultured Sertoli cells.

Within the human testis, large amounts of sulfated steroid hormones are produced. As shown in breast tissue and placenta, these might not only be excretion intermediates, but re-activated in target cells by steroid sulfatase (STS). This process is called sulfatase pathway and may play a pivotal role in para- and/or intracrine regulation by creating a local supply for steroid hormones. This requires a facilitated transport via uptake carriers and efflux transporters as these hydrophilic molecules cannot pass the cell membrane. Moreover, blood-testis barrier formation in the testis requires a transport through Sertoli cells (SCs) to reach germ cells (GCs). Sertoli cells are therefore expected to play a key role as gate-keepers for sulfatase pathway in human seminiferous epithelium. We analyzed the mRNA and protein expression of uptake carriers and efflux transporters like organic anion-transporting polypeptides (OATP2B1, OATP3A1) and multidrug resistance-related proteins (MRP1, MRP4) in testicular tissue and cultured Sertoli cells (FS1, HSEC). Additionally, expression pattern of STS as well as sulfonating enzymes (SULTs) were assessed. OATP2B1, OATP3A1 and STS were detected in SCs as well as GCs, whereas MRP1 is only expressed in SCs, and SULT1E1 only in Leydig cells, respectively. By transcellular transport of [H3]DHEAS in HSEC, we showed a functional transport of sulfated steroids in vitro. Our data indicate that steroid synthesis via sulfatase pathway in Sertoli cells in vivo and in vitro is possible and may contribute to paracrine and intracrine regulation employing the local supply of sulfated and free steroid hormones inside seminiferous tubules.

Transfection of Sertoli cells with androgen receptor alters gene expression without androgen stimulation.

BACKGROUND: Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is involved in various cell biological processes such as sex differentiation. To study the functional mechanisms of androgen action, cell culture systems and AR-transfected cell lines are needed. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. However, it remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. Therefore, we transfected Ar-deficient rat Sertoli cells 93RS2 by electroporation using a full length human AR. RESULTS: Transfection success was confirmed by Western Blotting, immunofluorescence and RT-PCR. AR transfection-related gene expression alterations were detected with microarray-based genome-wide expression profiling of transfected and non-transfected 93RS2 cells without androgen stimulation. Microarray analysis revealed 672 differentially regulated genes with 200 up- and 472 down-regulated genes. These genes could be assigned to four major biological categories (development, hormone response, immune response and metabolism). Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes. CONCLUSION: We conclude from our data, that the transfection of Ar-deficient Sertoli cells with AR has a measurable effect on gene expression even without androgen stimulation and cause Sertoli cell damage. Studies using AR-transfected cells, subsequently stimulated, should consider alterations in AR-dependent gene expression as off-target effects of the AR transfection itself.

Tenogenic differentiation of equine adipose-tissue-derived stem cells under the influence of tensile strain, growth differentiation factors and various oxygen tensions.

Mesenchymal stem cells have become extremely interesting for regenerative medicine and tissue engineering in the horse. Stem cell therapy has been proven to be a powerful and successful instrument, in particular for the healing of tendon lesions. We pre-differentiated equine adipose-tissue-derived stem cells (ASCs) in a collagen I gel scaffold by applying tensile strain, growth differentiation factors (GDFs) and various oxygen tensions in order to determine the optimal conditions for in vitro differentiation toward the tenogenic lineage. We compared the influence of 3% versus 21% oxygen tension, the use of GDF 5, GDF 6 and GDF 7 and the application of uniaxial tensile strain versus no mechanical stimulation on differentiation results as evaluated by cell morphology and by the expression of the tendon-relevant genes collagen I, collagen III, cartilage oligomeric matrix protein and scleraxis. The best results were obtained with an oxygen tension of 21%, tensile stimulation and supplementation with GDF 5 or GDF 7. This approach raises the hope that the in vivo application of pre-differentiated stem cells will improve healing and recovery time in comparison with treatment involving undifferentiated stem cells.

Identification of gonadal tissue in cryptorchid stallion can be improved by molecular biological analysis - a case report.

Surgically removed testicular tissue in cryptorchid stallions is sometimes difficult to identify because of morphological and histological malformation. Therefore, a sure method to characterise the removed tissue is required. A 2-year-old Haflinger stallion was castrated after diagnosis of cryptorchidism to remove the left intra-abdomnial testis. Intra-operative exploration of the abdominal cavity revealed a firm, dysmorphic structure, which could not be identified as testis based on macroscopic anatomy. The removed tissue was Bouin-fixed and paraffin-embedded for histological examination. We additionally applied immuno-histochemistry for smooth muscle actin to identify tubular structures as well as reverse transcriptase polymerase chain reaction (RT-PCR) to detect the expression of steroidogenic acute regulatory protein (StAR), indicating the presence of Leydig cells. A hCG test was conducted after surgery to screen for remaining testicular tissue. Histological examination using haematoxylin and eosin staining revealed signs of tissue calcification, amorph matrix with scattered cells and round structures. The latter could not be definitely identified as tubules. Actin staining revealed a few tubular wall structures. StAR mRNA expression indicated the presence of Leydig cells in parts of the removed tissue. The hCG test after castration showed no increase in testosterone. Histological and molecular biological examination of extirpated tissue in cryptorchid stallions can play an important role in the identification of the malformed testes like structures. The use of molecular biological techniques provides the opportunity to characterise surgically removed abdominal tissue that cannot be clearly diagnosed by routine histological examination.

Translational control of the AZFa gene DDX3Y by 5'UTR exon-T extension.

The human DEAD-box Y (DBY) RNA helicase (aka DDX3Y) gene is thought to be the major azoospermia factor a (AZFa) gene in proximal Yq11. Although it is transcribed in many tissues, the protein is expressed only in spermatogonia. In this study, we demonstrate that this translational control mechanism is probably germ cell-specific because of its association with expression of a distinct class of DDX3Y testis transcripts present only in pre- and post-meiotic male germ cells. They are initiated from a second distal DDX3Y promoter domain at two distinct start sites in the gene's 5' untranslated region (UTR) exon-T sequence. With the aid of an EGFP-3xFLAG reporter cassette cloned downstream of DDX3Y minigenes containing exons 1-4 and two different exon-T extensions, we discovered that DDX3Y translation is influenced by the presence of several ATG triplets located in exon-T, thus upstream of the main translational ATG start codon in exon 1. Strong translational repression of the DDX3Y minigene transcripts was observed when they contained the longest exon-T sequence with five upstream ATG triplets (uATGs). The potential formation of complex distinct stem-loop structures serve here as additional repressor element. Only minor translational attenuation was seen for the DDX3Y minigene transcripts when containing the shortest exon-T sequence, that is, starting at first transcriptional start site (coined 'T-TSS-I'). It was completely released after its single uATG was abolished by mutation. As we found DDX3Y transcripts with the longest exon-T sequence predominantly in spermatids, our results suggest that the amount of DDX3Y protein in pre-meiotic germ cells and its absence in post-meiotic germ cells are tightly controlled by the different extensions of exon-T in this germ cell-specific DDX3Y transcript class.