Adult Germ Cells Research Articles

PiRNAs Power Sperm Development in the Adult

PIWI-interacting RNAs (piRNAs)—small 23–32 nucleotide single-stranded RNAs—function before birth in prospermatogonia, the early cellular precursors to mammalian sperm. There, piRNAs are known to quell the activity of transposable elements. But another set of piRNAs operates in the adult testis, and less is known about what these RNAs do. A new study finds that these RNAs not only quell transposable elements in the testis, they also seem to regulate the developmental program of spermatogenesis, possibly by altering chromatin state [1]. Shu Ly Lim et al. began by asking about methylation of piRNAs, which is known to occur in the testis at the 3 end of these RNAs. In other species, the enzyme that mediates this methylation has been identified, HEN methyltransferase (HENMT1). To explore the role of this enzyme in mammals, the researchers examined male mice homozygous for a point mutation in the gene encoding HENMT1. The researchers found that piRNAs in the testis were unmethylated and unstable. In addition, these males were infertile, with a range of defects in sperm development in the testis. Retrotransposons became active in adult meiotic cells and haploid germ cells, and many RNAs involved in spermatogenesis were precociously expressed. It seems as if the testis-specific piRNAs do not operate by affecting RNA stability or by regulating translation, tasks that small RNAs are known to do in other cells. Instead, the researchers provide evidence that these piRNAs—also called pachytene piRNAs—can modify gene expression by altering chromatin. The researchers performed ChIP and and qPCR experiments on several key spermiogenesis genes in wild-type and mutant mice. The genes in the mutant mice contained increased levels of activating histone marks, suggestive of an open, transcriptionally permissive chromatin structure. This finding jibes with previous studies in flies, showing that piRNAs affect histone marks. The phenotype of the HENMT1 mutant mice resembles a human infertility condition called oligoasthenoteratospermia (OAT), which is treated with ICSI (intra-cytoplasmic sperm injection). The researchers raise the concern that such individuals may be transmitting sperm with active transposable elements and massively altered mRNA profiles, perhaps to the detriment of their descendants’ health.

Mechano-logical model of C. elegans germ line suggests feedback on the cell cycle

The Caenorhabditis elegans germ line is an outstanding model system in which to study the control of cell division and differentiation. Although many of the molecules that regulate germ cell proliferation and fate decisions have been identified, how these signals interact with cellular dynamics and physical forces within the gonad remains poorly understood. We therefore developed a dynamic, 3D in silico model of the C. elegans germ line, incorporating both the mechanical interactions between cells and the decision-making processes within cells. Our model successfully reproduces key features of the germ line during development and adulthood, including a reasonable ovulation rate, correct sperm count, and appropriate organization of the germ line into stably maintained zones. The model highlights a previously overlooked way in which germ cell pressure may influence gonadogenesis, and also predicts that adult germ cells might be subject to mechanical feedback on the cell cycle akin to contact inhibition. We provide experimental data consistent with the latter hypothesis. Finally, we present cell trajectories and ancestry recorded over the course of a simulation. The novel approaches and software described here link mechanics and cellular decision-making, and are applicable to modeling other developmental and stem cell systems.

Cell cycle features of C. elegans germline stem/progenitor cells vary temporally and spatially

Many organisms accumulate a pool of germline stem cells during development that is maintained in later life. The dynamics of establishment, expansion and homeostatic maintenance of this pool are subject to both developmental and physiological influences including the availability of a suitable niche microenvironment, nutritional status, and age. Here, we investigated the dynamics of germline proliferation during stages of expansion and homeostasis, using the C. elegans germ line as a model. The vast majority of germ cells in the proliferative zone are in interphase stages of mitosis (G1, S, G2) rather than in the active mitotic (M) phase. We examined mitotic index and DNA content, comparing different life stages, mutants, and physiological conditions. We found that germ cells in larval stages cycle faster than in adult stages, but that this difference could not be attributed to sexual fate of the germ cells. We also found that larval germ cells exhibit a lower average DNA content compared to adult germ cells. We extended our analysis to consider the effects of distance from the niche and further found that the spatial pattern of DNA content differs between larval and adult stages in the wild type and among mutants in pathways that interfere with cell cycle progression, cell fate, or both. Finally, we characterized expansion of the proliferative pool of germ cells during adulthood, using a regeneration paradigm (ARD recovery) in which animals are starved and re-fed. We compared adult stage regeneration and larval stage expansion, and found that the adult germ line is capable of rapid accumulation but does not sustain a larval-level mitotic index nor does it recapitulate the larval pattern of DNA content. The regenerated germ line does not reach the number of proliferative zone nuclei seen in the continuously fed adult. Taken together, our results suggest that cell cycle dynamics are under multiple influences including distance from the niche, age and/or maturation of the germ line, nutrition and, possibly, latitude for physical expansion.

Sphere-formation culture of testicular germ cells in the common marmoset, a small New World monkey.

Spermatogonia are specialized cells responsible for continuous spermatogenesis and the production of offspring. Because of this biological property, in vitro culture of spermatogonia provides a powerful methodology to advance reproductive biology and engineering. However, methods for culturing primate spermatogonia are poorly established. We have designed a novel method for culturing spermatogonia in the common marmoset (Callithrix jacchus), a small primate. By using our method with a suite of growth factors, adult marmoset testis-derived germ cells could be cultured in the form of a floating sphere for several weeks. Notably, this method could be applied not only to freshly isolated cells but also to cryopreserved cell stocks. The spheres enriched spermatogonia and early spermatocytes, and could be assembled from a C-KIT(+) spermatogonial population. Techniques for culturing spermatogonia could facilitate increased understanding of primate reproduction as well as the preservation of valuable biomaterials from nonhuman primates.

Chromosome 12p abnormalities and IMP3 expression in prepubertal pure testicular teratomas

Although the histologic appearance of pure testicular teratomas (PTTs) is similar in children and adults, the prognosis is dramatically different. Prepubertal PTTs are rare, with a benign clinical course, whereas the adult cases typically have malignant outcomes. Chromosome 12p abnormalities are seen in most adult testicular germ cell tumors but have not been found in prepubertal PTTs. IMP3 is an oncofetal protein that is highly expressed in many malignancies. Recently, we demonstrated IMP3 is expressed in adult mature testicular teratomas but not in mature ovarian teratomas. The aim of this study was to evaluate prepubertal PTTs for chromosome 12p abnormalities and expression of IMP3. A total of 11 cases (excision, n=1; orchiectomy, n=10) were obtained from the surgical pathology archives of 2 large medical centers (1957-2013). All 11 cases were investigated for isochromosome 12p and 12p copy number gain using interphase fluorescence in situ hybridization analysis and were examined by immunohistochemistry for IMP3 expression. Patients ranged in age from 0.9 to 7.0 (mean, 2.4) years. A positive immunohistochemical stain for IMP3 (cytoplasmic staining) was identified in 5 (46%) of 11 cases. Isochromosome 12p was detected in 2 cases (18%) that also expressed IMP3. Somatic copy number alterations of 12p were not observed (0%). We are the first to describe 12p abnormalities and IMP3 expression in prepubertal PTTs. Our data demonstrate a small subset of PTTs harbor typical molecular alterations observed in adult testicular germ cell tumors. Although prepubertal PTTs are considered to be benign neoplasms, it may be a heterogeneous group.

HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse.

piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2’ O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program.

Diagnostic markers for germ cell neoplasms: from placental-like alkaline phosphatase to micro-RNAs.

This concise review summarises tissue and serum markers useful for differential diagnosis of germ cell tumours (GCT), with focus on the most common testicular GCT (TGCT). GCT are characterised by phenotypic heterogeneity due to largely retained embryonic pluripotency and aberrant somatic differentiation. TGCT that occur in young men are divided into two main types, seminoma and nonseminoma, both derived from a pre-invasive germ cell neoplasia in situ (GCNIS), which originates from transformed foetal gonocytes. In severely dysgenetic gonads, a GCNIS-resembling lesion is called gonadoblastoma. GCT occur rarely in young children (infantile GCT) in whom the pathogenesis is different (no GCNIS/gonadoblastoma stage) but the histopathological features are similar to the adult GCT. The rare spermatocytic tumour of older men is derived from post-pubertal spermatogonia that clonally expand due to gain-of function mutations in survival-promoting genes (e.g. FGFR3, HRAS), thus this tumour has a different expression profile than GCNIS-derived TGCT. Clinically most informative immunohistochemical markers for GCT, except teratoma, are genes expressed in primordial germ cells/gonocytes and embryonic pluripotency-related factors, such as placental-like alkaline phosphatase (PLAP), OCT4 (POU5F1), NANOG, AP-2γ (TFAP2C) and LIN28, which are not expressed in normal adult germ cells. Some of these markers can also be used for immunocytochemistry to detect GCNIS or incipient tumours in semen samples. Gene expression in GCT is regulated in part by DNA and histone modifications, and the epigenetic profile of these tumours is characterised by genome-wide demethylation, except nonseminomas. In addition, a recently discovered mechanism of post-genomic gene expression regulation involves small non-coding RNAs, predominantly micro-RNA (miR). Testicular GCT display micro-RNA profiles similar to embryonic stem cells. Targeted miRNA-based blood tests for miR-371-3 and miR-367 clusters are currently under development and hold a great promise for the future. In some patients miR-based tests may be even more sensitive than the classical serum tumour markers, β -chorio-gonadotrophin (β-hCG), α-fetoprotein (AFP) and lactate dehydrogenase (LDH), which are currently used in the clinic. In summary, research advances have provided clinicians with a panel of molecular markers, which allow specific diagnosis of various subtypes of GCT and are very useful for early detection at the precursor stage and for monitoring of patients during the follow-up.

The value of positive Oct3/4 and D2-40 immunohistochemical expression in prediction of germ cell neoplasia in prepubertal boys with cryptorchidism

Objective: Intratubular germ cell neoplasia (ITGCN) is a precursor to testicular germ cell cancer. Adult germ cell cancer immunohistochemical markers fail to detect ITGCN in prepubertal boys with congenital cryptorchidism, because positive immunohistochemistry is commonly seen below 18 months old, where most orchiopexies are performed. The aim of the study was to evaluate the ability of Oct3/4 and D2-40 immunohistochemical markers to detect ITGCN in boys older than 2 years with cryptorchidism. Materials and methods: Histological sections from 309 testicular biopsies from 234 boys aged 1 month to 14 years, 6 months operated on for cryptorchidism were incubated with primary antibodies including anti-placental-like alkaline phosphatase, anti-Oct3/4, anti-C-kit and anti-D2-40 receptor. Results: One 3-year, 8-month-old boy with 45X/46XY disorder of sexual development had ITGCN and all positive markers. Besides this case, none of the 192 testes except one from boys older than 2 years had any Oct3/4- or D2-40-positive germ cells identified. The germ cells of the right testis from a 3-year, 7-month-old boy had weak Oct3/4 expression but were D2-40 negative. The prevalences of Oct3/4- and D2-40-positive staining of germ cells in testicular biopsies were, for each age group: < 6 months, 100% and 50%; 6 months to < 1 year, 65% and 16%; 1 to < 2 years, 15% and 3%; and 2 years to < 14 years, 6 months, 2% and 1%, respectively. Conclusion: Oct3/4 and D2-40 immunohistochemical markers may be beneficial in detecting ITGCN in boys older than 2 years with cryptorchidism. Even when immunohistochemistry is applied, prepubertal ITGCN is so rarely demonstrated in cryptorchid testes that it is not plausible that ITGCN generally originates during fetal development in cryptorchidism.

Effects of paracrine factors on CD24 expression and neural differentiation of male germline stem cells.

Spermatogonial stem cells (SSCs) are adult male germ cells that develop after birth. Throughout the lifetime of an organism, SSCs sustain spermatogenesis through self-renewal and produce daughter cells that differentiate into spermatozoa. Several studies have demonstrated that SSCs can acquire pluripotency under appropriate culture conditions, thus becoming multipotent germline stem cells (mGSCs) that express markers of pluripotency in culture and form teratomas following transplantation into immunodeficient mice. In the present study, we generated neural precursor cells expressing CD24, a neural precursor marker, from pluripotent stem cell lines and demonstrated that these cells effectively differentiated along a neural lineage in vitro. In addition, we found that paracrine factors promoted CD24 expression during the neural differentiation of mGSCs. Our results indicated that the expression of CD24, enhanced by a combination of retinoic acid (RA), noggin and fibroblast growth factor 8 (FGF8) under serum-free conditions promoted neural precursor differentiation. Using a simple cell sorting method, we were able to collect neural precursor cells with the potential to differentiate from mGSCs into mature neurons and astrocytes in vitro.

Interrogation of a context-specific transcription factor network identifies novel regulators of pluripotency.

The predominant view of pluripotency regulation proposes a stable ground state with coordinated expression of key transcription factors (TFs) that prohibit differentiation. Another perspective suggests a more complexly regulated state involving competition between multiple lineage-specifying TFs that define pluripotency. These contrasting views were developed from extensive analyses of TFs in pluripotent cells in vitro. An experimentally validated, genome-wide repertoire of the regulatory interactions that control pluripotency within the in vivo cellular contexts is yet to be developed. To address this limitation, we assembled a TF interactome of adult human male germ cell tumors (GCTs) using the Algorithm for the Accurate Reconstruction of Cellular Pathways (ARACNe) to analyze gene expression profiles of 141 tumors comprising pluripotent and differentiated subsets. The network (GCT(Net)) comprised 1,305 TFs, and its ingenuity pathway analysis identified pluripotency and embryonal development as the top functional pathways. We experimentally validated GCT(Net) by functional (silencing) and biochemical (ChIP-seq) analysis of the core pluripotency regulatory TFs POU5F1, NANOG, and SOX2 in relation to their targets predicted by ARACNe. To define the extent of the in vivo pluripotency network in this system, we ranked all TFs in the GCT(Net) according to sharing of ARACNe-predicted targets with those of POU5F1 and NANOG using an odds-ratio analysis method. To validate this network, we silenced the top 10 TFs in the network in H9 embryonic stem cells. Silencing of each led to downregulation of pluripotency and induction of lineage; 7 of the 10 TFs were identified as pluripotency regulators for the first time.

Cloning Efficiency and a Comparison between Donor Cell Types

Dolly the sheep cloning “the first cloning process” was derived by somatic cell nuclear transfer and it had reported that the clone got premature senescence as a result of continues telomere shortening and ongoing decrease in telomerase activity by time; thus majority of recent researches is directed to use other types of donor cells to be transferred into the recipient enucleated oocyte to maintain full-term and healthy postnatal development for the clone. Embryonic cells such as embryonic stem cell and primordial germ cell were on top as a product of high activity of telomerase, but it cannot be used to clone an adult and primordial germ cell could only be used in nuclear transfer in early stages of embryonic development. Adult stem cell and adult germ cell also had shown increase in telomerase activity but using germ cell as a donor cell at specific day postcoitum had shown incompetence to satisfy full-term development of embryo making it inadequate to be donor cell in cloning process. Another effective cell that could reverse cellular aging in cloning is fibroblast cell. Sertoli cell in male and cumulus cell in female had shown the high efficiency to be donor cells in nuclear transfer. As we suppose that cloning will soon be applicable in many fields such as pharmacy, agriculture, biotechnology and medicine, here, we review current progress in cloning including several types of donor cells had been used in cloning.

Mechano-logical model of C. elegans germ line suggests feedback on the cell cycle

The Caenorhabditis elegans germ line is an outstanding model system in which to study the control of cell division and differentiation. Although many of the molecules that regulate germ cell proliferation and fate decisions have been identified, how these signals interact with cellular dynamics and physical forces within the gonad remains poorly understood. We therefore developed a dynamic, 3D in silico model of the C. elegans germ line, incorporating both the mechanical interactions between cells and the decision-making processes within cells. Our model successfully reproduces key features of the germ line during development and adulthood, including a reasonable ovulation rate, correct sperm count, and appropriate organization of the germ line into stably maintained zones. The model highlights a previously overlooked way in which germ cell pressure may influence gonadogenesis, and also predicts that adult germ cells might be subject to mechanical feedback on the cell cycle akin to contact inhibition. We provide experimental data consistent with the latter hypothesis. Finally, we present cell trajectories and ancestry recorded over the course of a simulation. The novel approaches and software described here link mechanics and cellular decision-making, and are applicable to modeling other developmental and stem cell systems.

GLD-4-mediated translational activation regulates the size of the proliferative germ cell pool in the adult C. elegans germ line.

To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.

Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations.

Germ cells are maintained in a pristine non-aging state as they proliferate over generations. Here, we show that a novel function of the Caenorhabditis elegans RNA interference proteins RNAi spreading defective (RSD)-2 and RSD-6 is to promote germ cell immortality at high temperature. rsd mutants cultured at high temperatures became progressively sterile and displayed loss of small interfering RNAs (siRNAs) that target spermatogenesis genes, simple repeats, and transposons. Desilencing of spermatogenesis genes occurred in late-generation rsd mutants, although defective spermatogenesis was insufficient to explain the majority of sterility. Increased expression of repetitive loci occurred in both germ and somatic cells of late-generation rsd mutant adults, suggesting that desilencing of many heterochromatic segments of the genome contributes to sterility. Nuclear RNAi defective (NRDE)-2 promotes nuclear silencing in response to exogenous double-stranded RNA, and our data imply that RSD-2, RSD-6, and NRDE-2 function in a common transgenerational nuclear silencing pathway that responds to endogenous siRNAs. We propose that RSD-2 and RSD-6 promote germ cell immortality at stressful temperatures by maintaining transgenerational epigenetic inheritance of endogenous siRNA populations that promote genome silencing.

Peritubular myoid cells participate in male mouse spermatogonial stem cell maintenance.

Peritubular myoid (PM) cells surround the seminiferous tubule and together with Sertoli cells form the cellular boundary of the spermatogonial stem cell (SSC) niche. However, it remains unclear what role PM cells have in determining the microenvironment in the niche required for maintenance of the ability of SSCs to undergo self-renewal and differentiation into spermatogonia. Mice with a targeted disruption of the androgen receptor gene (Ar) in PM cells experienced a progressive loss of spermatogonia, suggesting that PM cells require testosterone (T) action to produce factors influencing SSC maintenance in the niche. Other studies showed that glial cell line-derived neurotrophic factor (GDNF) is required for SSC self-renewal and differentiation of SSCs in vitro and in vivo. This led us to hypothesize that T-regulated GDNF expression by PM cells contributes to the maintenance of SSCs. This hypothesis was tested using an adult mouse PM cell primary culture system and germ cell transplantation. We found that T induced GDNF expression at the mRNA and protein levels in PM cells. Furthermore, when thymus cell antigen 1-positive spermatogonia isolated from neonatal mice were cocultured with PM cells with or without T and transplanted to the testes of germ cell-depleted mice, the number and length of transplant-derived colonies was increased considerably by in vitro T treatment. These results support the novel hypothesis that T-dependent regulation of GDNF expression in PM cells has a significant influence on the microenvironment of the niche and SSC maintenance.

Differential expression of boule and dazl in adult germ cells of the Asian seabass

Fertility genes boule and dazl constitute the evolutionarily conserved DAZ (Deleted in AZoospermia) family of RNA binding proteins essential for germline development across animal phyla. Here we report the cloning and expression analysis of boule and dazl from the Asian seabass (Lates calcarifer), a marine fish that undergoes sequential male-to-female sex reversal. Molecular cloning and sequence comparison led to the identification of boule and dazl cDNAs. RT-PCR analysis showed that both boule and dazl RNAs were restricted to the gonads among adult organs examined. Chromogenic in situ hybridization revealed germ cell-specific expression for both boule and dazl in female and male adults. Importantly, distinct differences were found between boule and dazl in terms of temporospatial expression and subcellular distribution. The boule RNA was abundant in late gametogenic cells except sperm. Interestingly, dazl expression increases in early oocytes and concentrates in a perinuclear speckle that appears to develop ultimately into the Balbiani body in advanced oocytes. The dazl RNA was found to be abundant in spermatocytes but hardly detectable in sperm. These data demonstrate that boule and dazl are germ cell markers in the adult Asian seabass, and that bisexual germline-specific expression has been conserved for boule and dazl in fish.

Differential expression of IMP3 between male and female mature teratomas—immunohistochemical evidence of malignant nature

Ovarian mature teratoma is a benign tumour, whereas mature teratoma in adult testicular germ cell tumours (TGCTs) is considered to be a malignant tumour. IMP3, an oncofetal protein, plays an important role in embryogenesis and carcinogenesis. IMP3 has been demonstrated to be a malignant biomarker that is mainly expressed in malignant neoplasms rather than benign tissues. The aim of this study was to analyse IMP3 expression in germ cell tumours, and compare its expression between male and female teratomas. One hundred and seventy-eight cases (62 TGCTs, 52 ovarian teratomas, 27 metastatic testicular teratomas, and 37 cases of normal testicular tissue) obtained from the archives of two large academic medical centres were examined for IMP3 expression. Of the 62 TGCTs, 30 had mature teratoma components. IMP3 expression was present in 100% (30/30) of testicular mature teratoma components, and in 96% (26/27) of metastatic testicular teratomas. Other TGCT components also expressed IMP3 in 99% of cases (78/79). IMP3 expression was negative in all female mature teratomas. We describe for the first time an immunostaining marker that has differential expression in male and female mature teratomas, indicating that their pathogenesis differs. High expression of IMP3 in adult mature testicular teratomas supports their malignant nature.

Light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka.

Germ cells of diverse animal species have a unique membrane-less organelle called germ plasm (GP). GP is usually associated with mitochondria and contains RNA binding proteins and mRNAs of germ genes such as vasa. GP has been described as the mitochondrial cloud (MC), intermitochondrial cement (IC) and chromatoid body (CB). The mechanism underlying varying GP structures has remained incompletely understood. Here we report the analysis of GP through light and electron microscopy by using Vasa as a marker in adult male germ cells of the fish medaka (Oryzias latipes). Immunofluorescence light microscopy revealed germ cell-specific Vasa expression. Vasa is the most abundant in mitotic germ cells (oogonia and spermatogonia) and reduced in meiotic germ cells. Vasa in round spermatids exist as a spherical structure reminiscent of CB. Nanogold immunoelectron microscopy revealed subcellular Vasa redistribution in male germ cells. Vasa in spermatogonia concentrates in small areas of the cytoplasm and is surrounded by mitochondria, which is reminiscent of MC. Vasa is intermixed with mitochondria to form IC in primary spermatocytes, appears as the free cement (FC) via separation from mitochondria in secondary spermatocyte and becomes condensed in CB at the caudal pole of round spermatids. During spermatid morphogenesis, Vasa redistributes and forms a second CB that is a ring-like structure surrounding the dense fiber of the flagellum in the midpiece. These structures resemble those described for GP in various species. Thus, Vasa identifies GP and adopts varying structures via dynamic reorganization at different stages of germ cell development.

Validation of endogenous normalizing genes for expression analyses in adult human testis and germ cell neoplasms.

The measurement of gene expression levels in cells and tissues typically depends on a suitable point of reference for inferring biological relevance. For quantitative (or real-time) RT-PCR assays, the method of choice is often to normalize gene expression data to an endogenous gene that is stably expressed across the samples analysed: a so-called normalizing or housekeeping gene. Although this is a valid strategy, the identification of stable normalizing genes has proved challenging and a gene showing stable expression across all cells or tissues is unlikely to exist. Therefore, it is necessary to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers, highlighting the importance of selecting appropriate normalizing genes as comparative measurements can yield variable results when different normalizing genes are employed. Based on our results, we recommend using RPS20, RPS29 or SRSF4 when analysing relative gene expression levels in human testis and associated testicular pathologies. OCT4 and SALL4 can be used with caution as second-tier normalizers when determining changes in gene expression in germ cells and germ cell tumour components, but the relative transcript abundance appears variable between different germ cell tumour types. We further recommend that such studies should be accompanied by additional assessment of histology and cellularity of each sample.

Effect of advancing paternal age on semen parameters and seminal oxidative stress markers in infertile men

BackgroundHigh rate of subfertility and adverse pregnancy related out-comes associated with childbearing are seen after age 40.In contrast to oogenesis, spermatogenesis continues inelderly men. Adult male germ cells pass through signifi-cantly more mitotic replications than the germ cells inadult female. In men, there is an age associated increase inthe incidence of breaks in sperm DNA, decrease in apopto-sis, and a higher frequency of point mutations. Advancedpaternal age is associated with an increased time to preg-nancy and decreased pregnancy rates. After adjusting forfemale age, conception during a 12-month period was > 30percent less likely for men over 40 years of age as com-pared to men 45years compared to men 40 years (n = 105). We evaluated the conven-tional semen parameters (WHO, 2010) [5] and OS mar-kers: seminal ROS (chemiluminescence assay), totalantioxidant capacity (TAC) and sperm DNA damage(TUNEL assay) in all patients.ResultsThe mean age of the study subjects was 36.8 ± 6.7 years.No age-related differences were seen in conventionalsemen parameters (volume, concentration, motility, andmorphology) (Table 1). ROS and antioxidant levels werecomparable in the 3 groups. Significantly higher levels ofsperm DNA damage (19.94 ± 15.30%) was seen in infertilemen >40 years compared to men in younger age groups(P = 0.028 and P = 0.027, respectively).ConclusionsSperm DNA damage increases with advancing paternalage. Evaluation of sperm DNA damage will help diagnosethe underlying cause of poor fertility in some men andassist the clinician in offering correct treatment modality.