Germ Cell Maturation Research Articles

The potential function of prohibitin during spermatogenesis in Chinese fire-bellied newt Cynops orientalis.

Prohibitin proteins are multifunctional proteins located mainly at the inner membrane of mitochondria expressed in universal species. They play a vital role in mitochondria's function, cell proteolysis, senescence, apoptosis and as a substrate for ubiquitination. In this study, we used PCR cloning, protein and nucleotide acids alignment, protein structure prediction, western blot, in situ hybridization and immunofluorescence to study the characteristics of the prohibitin gene and the potential role of prohibitin in spermatogenesis and spermiogenesis processes in the Chinese fire-bellied newt Cynops orientalis. First, we cloned a 1452-bp full-length cDNA from the testis of Cynops orientalis. Second, we found that the 272 amino acids of prohibitin have a SPFH family domain. Thirdly, the western blots showed high expression of prohibitin in testis while the protein size was approximately 32kDa. Fourthly, the results of in situ hybridization and immunofluorescence experiments showed that most of the prohibitins travelled with the mitochondria's migration in Cynops orientalis. The quantities of mRNA decreased as spermiogenesis proceeded, although the signals of prohibitins existed during the whole period of spermatogenesis and spermiogenesis. In the mature germ cells, the signals of prohibitins were weak and aggregated at the end of the cell. Finally, we discovered that the Sertoli cells had a large quantity of prohibitins and we made several assumptions of prohibitins' potential roles in those cells. This is the first time that the relationship between mitochondria and prohibitin in different stages of the sperm cells in Cynops orientalis has been examined, which also revealed that Sertoli cells have abundant prohibitins.

Regulation of germ cell function by SUMOylation.

Oogenesis and spermatogenesis are tightly regulated complex processes that are critical for fertility. Germ cells undergo meiosis to generate haploid cells necessary for reproduction. Errors in meiosis, including the generation of chromosomal abnormalities, can result in reproductive defects and infertility. Meiotic proteins are regulated by post-translational modifications including SUMOylation, the covalent attachment of small ubiquitin-like modifier (SUMO) proteins. Here, we review the role of SUMO proteins in controlling germ cell development and maturation based on recent findings from mouse models. Several studies have characterized the localization of SUMO proteins in male and female germ cells. However, a deeper understanding of how SUMOylation regulates proteins with essential roles in oogenesis and spermatogenesis will provide useful insight into the underlying mechanisms of germ cell development and fertility.

Aquaporin biology of spermatogenesis and sperm physiology in mammals and teleosts.

Fluid homeostasis is recognized as a critical factor during the development, maturation, and function of vertebrate male germ cells. These processes have been associated with the presence of multiple members of the aquaporin superfamily of water and solute channels in different cell types along the reproductive tract as well as in spermatozoa. We present a comparative analysis of the existing knowledge of aquaporin biology in the male reproductive tissues of mammals and teleosts. Current data suggest that in both vertebrate groups, aquaporins may have similar functions during differentiation of spermatozoa in the germinal epithelium, in the concentration and maturation of sperm in the testicular ducts, and in the regulation of osmotically induced volume changes in ejaculated spermatozoa. Recent studies have also provided insight into the possible function of aquaporins beyond water transport, such as in signaling pathways during spermatogenesis or the sensing of cell swelling and mitochondrial peroxide transport in activated sperm. However, an understanding of the specific physiological functions of the various aquaporins during germ cell development and sperm motility, as well as the molecular mechanisms involved, remains elusive. Novel experimental approaches need to be developed to elucidate these processes and to dissect the regulatory intracellular pathways implicated, which will greatly help to uncover the molecular basis of sperm physiology and male fertility in vertebrates.

Abstract 843: Imputation and meta-analysis of five genome-wide association studies identify multiple new loci associated with testicular germ cell tumor

Abstract Genome-wide association studies (GWAS) have already identified 21 genetic loci associated with testicular germ cell tumor (TGCT). Many of the loci contain biologically plausible genes that function in male germ cell maturation and differentiation, as well as KIT-MAPK signaling and chromosomal segregation. We recently formed the international TEsticular CAncer Consortium (TECAC), and pooled data from the three published and one unpublished GWAS (3,556 TGCT cases and 13,969 controls) to identify additional novel susceptibility loci. We imputed across the data set using the 1,000 Genomes Project version 3 and conducted a fixed effects meta-analysis, including the first analysis of the X chromosome. Eleven new loci achieved genome-wide significance level (p<5e-8), mapping to the following regions: 2q14.2, 3q26.2, 4q35.2, 7q36.3, 10q26.13, 15q21.3, 15q22.31, 16q24.2, 17q12, 19p12 and Xq28, most of which harbor biologically plausible genes. The per allele odds ratios associated with these SNPs continue to be higher than those associated with other cancer types, ranging from 1.2 to 1.6. The signal at 17q12 maps to a region which includes HNF1B, a locus already associated with risk of endometrial and prostate cancer. Two of the signals are in the introns of genes known to be involved in embryonal stem cell pluripotency (TFCLP1 - 2q14.2 and ZFPA2/REX1 - 4q35.2), one in the intron of a chromosomal segregation gene (NCAPG - 27q36.3), and one proximate to MEK1 (MAP2K1 - 15q22.31). The identification of these loci provides additional evidence of the importance of the previously implicated biological pathways. Heritability analysis indicated that these eleven new loci explain approximately 7% more familial (father-to-son) risk in addition to ∼26% explained by previously established 21 loci. Our new findings substantially increase the number of known TGCT susceptibility alleles, thus moving the field closer to a comprehensive understanding of the underlying genetic architecture of TGCT, and providing further clues into the biological etiology of TGCT. Citation Format: Zhaoming Wang, Peter A. Kanetsky, Katherine A. McGlynn, D. Timothy Bishop, Charles C. Chung, Marlene D. Dalgaard, Tom Grotmol, Mark H. Greene, Ramneek Gupta, Trine B. Haugen, Kevin Litchfield, Jennifer T. Loud, Nandita Mitra, Kasper Nielsen, Clare Turnbull, Ewa Rajpert-DeMeyts, Saran Vardhanabhuti, Fredrik Wiklund, Stephen Schwartz, Stephen J. Chanock, Katherine L. Nathanson, TECAC consortium. Imputation and meta-analysis of five genome-wide association studies identify multiple new loci associated with testicular germ cell tumor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 843. doi:10.1158/1538-7445.AM2015-843

WITHDRAWN: Comparative proteome analysis of human testis from newborn, young adult, and aged men identified spermatogenesis-associated proteins.

Ahead of Print article withdrawn by publisher.

ABO (histo) blood group phenotype development and human reproduction as they relate to ancestral IgM formation: A hypothesis

The formation of a histo (blood) group) ABO phenotype and the exclusion of an autoreactive IgM or isoagglutinin activity arise apparently in identical glycosylation of complementary domains on cell surfaces and plasma proteins. The fundamental O-glycan emptiness of the circulating IgM, which during the neonatal amino acid sequencing of the variable regions is exerting germline-specific O-GalNAc glycan-reactive serine/threonine residues that in the plasma of the adult human blood group O individuals apparently remain associated with the open glycosidic sites on the ABOH convertible red cell surface, must raise suggestions on a transient expression of developmental glycans, which have been “lost” over the course of maturation. In fact, while the mammalian non-somatic, embryogenic stem cell (ESC)- germ cell (GC) transformation is characterized by a transient and genetically as-yet-undefined trans-species-functional O-GalNAc glycan expression, in the C57BL/10 mouse such expression was potentially identified in growth-dependent, blood group A-like GalNAc glycan-bearing, ovarian glycolipids complementary with the syngeneic anti-A reactive IgM, which does not appear in early ovariectomized animals. This non-somatically encoded, polyreactive, ancestral IgM molecule has not undergone clonal selection and does primarily not differentiate between self and non-self and might, due to amino acid hydroxyl groups, highly suggest substrate competition with subsequent O-glycosylations in ongoing ESC-GC transformations and affecting GC maturation. However, the membrane-bound somatic N/O-glycotransferases, which initiate, after formation of the zygote, the complex construction of the human ABO phenotypes in the trans cisternae of the Golgi apparatus, are associated and/or completed with soluble enzyme versions exerting identical specificities in plasma and likely competing vice versa by glycosylation of neonatal IgM amino acids, where they suggest to accomplish the clearance of anti-A autoreactivity at germline serine and threonine residues. Sustaining the lineage-maintaining position of the classic A allele and the discovery of the OA hybrid alleles at the normal ABO locus and in heterozygous ESC lines have, together with clinical observations, raised discussions about a silent A-allelic support within blood group O reproduction. However, the question of whether a fictional “continued blood group O inbreeding” ultimately occurs without the A-allelic or somatic function remains unanswered because the genetic relationship between non-somatic O-GalNAc-glycosylations that operate before sperm-egg recognition and somatic O-GalNAc-glycosylations that arise after the formation of the zygote remains to be elucidated.

Retinoic acid treatment alters germ cell heterogeneity and testicular echotexture in prepubescent ram lambs.

Testicular echotextural attributes are closely associated with spermatogenic development; however, precise characterisation of specific germ cell types is difficult due to tremendous germ cell heterogeneity. Recently, retinoic acid (RA) administration in neonatal mice was found to induce highly synchronised spermatogenesis as adults. A RA-treatment protocol was tested in 17 ram lambs treated with or without RA at 8 weeks of age, with scrotal ultrasonography and blood samples collected until castration 24h or 2.5 weeks later. At 8.2 weeks of age, the nuclear:seminiferous tubule (ST) area was higher in the treated compared with the control group. Serum testosterone concentrations and numerical pixel values (NPVs) of the testicular parenchyma reached a peak at 9 weeks of age in both groups of ram lambs studied. At 10.5 weeks of age, the percentage of ST cross-sections with different germ cells as the most mature germ cell type was lower and the inter-tubular heterogeneity and NPVs were also lower in the treated compared with the control animals. RA manipulation of spermatogenesis in prepubertal ram lambs may provide a suitable model for further investigation of the echotextural characteristics of specific germ cell types and critical developmental events.

Characterization of Oct4-GFP transgenic mice as a model to study the effect of environmental estrogens on the maturation of male germ cells by using flow cytometry

Oct4 is involved in regulation of pluripotency during normal development and is down-regulated during formation of postnatal reservoir of germ cells. We propose thatOct4/GFP transgenic mouse, which mimics the endogenous expression pattern of Oct4, could be used as a mammalian model to study the effects of environmental estrogens on the development of male germ cells. Oct4/GFP maturation profile was assessed during postnatal days -PND- 3, 5, 7, 10, 14 and 80, using flow cytometry. Then, we exposed pregnant mothers to 17α-ethinylestradiol (EE2) from day post coitum (dpc) 5 to PND7. Percentage of Oct4/GFP-expressing cells and levels of expression of Oct4/GPF were increased in PND7 after EE2 exposure. These observations were confirmed by analysis of GFP and endogenous Oct4 protein in the seminiferous tubules and by a reduction in epididymal sperm count in adult mice. We introduced Oct4/GFP mouse together with flow cytometry as a tool to evaluate changes in male germ cells development.

Differential expression patterns of three aromatase genes and of four estrogen receptors genes in the testes of trout (Oncorhynchus mykiss).

Estrogens are implicated in male gonad function, although their physiological roles remain uncertain. In the present study, we take advantage of the original model of spatio-temporal organization of trout spermatogenesis to revisit the synthesis and action sites of estrogens in fish testis. Within this system, somatic cell and germ cell development are synchronized due to a strict seasonal spermatogenetic cycle and the cystic organization of gonads. We evaluated the expression patterns and regulation of three aromatase isoforms (cyp19a, cyp19b-I, and cyp19b-II) and four estrogen receptors (esr1a, esr1b, esr2a, and esr2b) by quantitative reverse-transcriptase PCR during testicular maturation and in isolated germ cell populations. Our data demonstrated a reciprocal relationship between cyp19a and cyp19b (I and II) expression during testicular development (cyp19a decreased while cyp19b increased with maturation). Furthermore, cyp19b is significantly expressed in late germ cells. At the protein level, aromatase was immunohistochemically identified in interstitial tissue and in germ cells. Remarkable elevation of esr1a and esr2a was observed during the final stage of spermiation, while esr1b was expressed in an early stage of spermatogenetic development. Estrogen implants reduced testicular cyp19a transcript abundance while up-regulating cyp19b levels, whereas androgens up-regulated testicular esr1a, esr2a, and esr2b. Together, the distinct spatio-temporal expression profiles and regulation of aromatases and estrogen receptors suggest that estrogens have discrete physiological functions during an early step of spermatogenesis and in the final stages of germ cell maturation and/or excretion.

The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells.

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.

Deficiency of spermatogenesis and reduced expression of spermatogenesis-related genes in prefoldin 5-mutant mice

MM-1α is a c-Myc-binding protein and acts as a transcriptional co-repressor in the nucleus. MM-1α is also PDF5, a subunit of prefoldin that is chaperon comprised of six subunits and prevents misfolding of newly synthesized nascent polypeptides. Prefoldin also plays a role in quality control against protein aggregation. It has been reported that mice harboring the missense mutation L110R of MM-1α/PFD5 exhibit neurodegeneration in the cerebellum and also male infertility, but the phenotype of infertility has not been fully characterized. In this study, we first analyzed morphology of the testis and epididymis of L110R of MM-1α mice. During differentiation of spermatogenesis, spermatogonia, spermatocytes and round spermatids were formed, but formation of elongated spermatids was compromised in L110R MM-1α mice. Furthermore, reduced number/concentration of sperm in the epididymis was observed. MM-1α was strongly expressed in the round spermatids and sperms with round spermatids, suggesting that MM-1α affects the differentiation and maturation of germ cells. Changes in expression levels of spermatogenesis-related genes in mice testes were then examined. The fatty-acid-binding protein (fabp4) gene was up-regulated and three genes, including sperm-associated glutamate (E)-rich protein 4d (speer-4d), phospholipase A2-Group 3 (pla2g3) and phospholipase A2-Group 10 (pla2g10), were down-regulated in L110R MM-1α mice. L110R MM-1α and wild-type MM-1α bound to regions of up-regulated and down-regulated genes, respectively. Since these gene products are known to play a role in maturation and motility of sperm, a defect of at least MM-1α transcriptional activity is thought to induce expressional changes of these genes, resulting in male infertility.

Genome wide DNA methylation profiles provide clues to the origin and pathogenesis of germ cell tumors.

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina’s HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809).

The influence of hormonal treatment with beta-human chorionic gonadotropin for cryptorchidism on future fertility in rats

There have been two treatment modalities for cryptorchidism such that surgical and hormonal; the latter being highly controversial. While some authors suggest that hormonal treatment increases the number and maturation of germ cells in cryptorchid testes, others believe just the opposite. We aimed to find out the sperm counts and testicular index; briefly fertility potential of the normally descended contralateral testes in adulthood period in rats treated with Beta-HCG in early period of their lives. Three groups, each including 10 rats aged 22 days old, in which delactation and normal feeding can be started, were formed to be Group 1: Sham operated, Group 2: Experimental cryptorchidism (EC) and Group 3: Hormone-treated after experimental cryptorchidism was performed (HT-EC). Left testis was placed in the abdomen in group 2 and 3. In group 1, a sham operation was performed. The rats in EC-HT group received subcutaneous injections of 50 IU/kg Beta-HCG daily for 7 days. Right orchidectomy was performed when they reached reproductive period to evaluate fertility potential with sperm counts and testicular index. Testicular index was calculated according to the formula "testicular length × width/weight of rat". Epididymal sperm count was made with hemocytometer. We evaluated the physical characteristics and fertility potential (sperm counts) of contralateral normal testes during adulthood in rats that underwent experimental unilateral cryptorchidism during infancy. A relationship between testis weight and sperm counts were also investigated. We could not find any direct correlation of sperm count with either testicular weight or testicular index in our study. Although the rats had normal testes at birth, we found decreased sperm counts in contralateral normal testes in EC group. This suggests that unilateral cryptorchidism may cause some systemic effects that reach the other testis. Hormone treatment was not beneficial. This is comparable to Nambirajan et al. who reported histological changes and decreased spermatogenic cell count in contralateral scrotal testes in experimentally induced unilateral cryptorchidism in early period of life in rats. Heiskanen et al. reported that treatment with Beta-HCG leads to decreased total sperm counts in the future due to increased germ cell apoptosis caused by hormonal withdrawal after treatment. Cortes et al. also reported decreased number of germ cells in 1-3 year-old boys who underwent surgery after unsuccessful Beta-HCG treatment. The reasons could be delayed testicular descent or adverse effect of hormone treatment though. Our results concurred with them. Apparently, our model has failed to mimic the pathophysiologic mechanisms of congenital cryptorchidism in humans. Furthermore, we applied hormone treatment in normal rats with normally descended testes. Therefore, the "by-product" information of our study is that, unnecessary use of Beta-HCG during infancy may impair future fertility. Our study suggests that Beta-HCG treatment may decrease sperm counts and decrease the future fertility potential. We could not find any direct correlation of sperm count with either testicular weight or testicular index.

Human Fetal Testicular Tissue Xenotransplantation: A Platform to Study the Effect of Gonadotropins on Human Germ Cell Development In Utero

We examined the effects of long-term hCG stimulation on germ cell maturation, and Sertoli and Leydig cell function in a xenotransplantation model of the human fetal testis. A total of 20 human fetal testes were ectopically xenografted on 20 castrated NCr male nude mice. Grafts were collected for analysis 24 weeks later. Mice were treated with saline as the control or with hCG beginning 4 weeks after the grafts were transplanted. Of the grafts 65% survived at 24 weeks. In contrast to untreated pregrafted samples, hCG stimulated xenografts showed significantly increased density of seminiferous tubule formation with Sertoli cell migration to the basement membrane. Germ cell proliferation and differentiation from gonocytes (M2A(+)) to prespermatogonia (MAGE-4A(+)) were observed in graft samples recovered from the hCG and nonhCG treated groups at 24 weeks of treatment. Leydig cells in hCG treated grafts produced significantly more testosterone than nonhCG treated grafts. Although further studies are required to investigate the potential for further differentiation and maturation of xenografted human fetal testes, normal in utero testicular development was reproduced under long-term hCG stimulation. This model represents a means to study long-term effects of gonadotoxins or hormonal stimulation on the maturation of human fetal testes.

NUT protein immunoreactivity in ovarian germ cell tumours

The aim of this study was to investigate NUT (nuclear protein in the testis) expression in ovarian germ cell tumours (GCTs). Immunostaining for NUT protein was performed in 10 mature cystic teratomas and in 49 malignant ovarian GCTs including 15 pure dysgerminomas, six dysgerminomas associated with gonadoblastoma, nine yolk sac tumours, 12 immature teratomas, and seven mixed malignant tumours. Only nuclear staining was considered a positive finding although cytoplasmic staining was noted when present. Thirty-seven (76%) malignant GCTs were NUT positive but staining was usually of weak to moderate intensity and observed in a relatively small proportion of neoplastic cells. Staining in immature teratomas and yolk sac tumours was restricted to foci of hepatoid and intestinal/glandular differentiation, where both nuclear and cytoplasmic reactivity were observed. In dysgerminoma associated with gonadoblastoma only the in situ and invasive germ cell elements were NUT positive. Nuclear staining was not seen in benign teratomas. Most malignant ovarian GCTs express NUT protein, albeit focally, and this should be considered when evaluating immunostaining in the differential diagnosis of poorly differentiated malignancies, particularly NUT midline carcinoma. Since NUT protein appears to play a role in normal germ cell maturation it may influence intestinal or hepatoid differentiation within malignant GCTs.

MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes.

The piRNA machinery is known for its role in mediating epigenetic silencing of transposons. Recent studies suggest that this function also involves piRNA-guided cleavage of transposon-derived transcripts. As many piRNAs also appear to have the capacity to target diverse mRNAs, this raises the intriguing possibility that piRNAs may act extensively as siRNAs to degrade specific mRNAs. To directly test this hypothesis, we compared mouse PIWI (MIWI)-associated piRNAs with experimentally identified cleaved mRNA fragments from mouse testes, and observed cleavage sites that predominantly occur at position 10 from the 5′ end of putative targeting piRNAs. We also noted strong biases for U and A residues at nucleotide positions 1 and 10, respectively, in both piRNAs and mRNA fragments, features that resemble the pattern of piRNA amplification by the 'ping-pong' cycle. Through mapping of MIWI-RNA interactions by CLIP-seq and gene expression profiling, we found that many potential piRNA-targeted mRNAs directly interact with MIWI and show elevated expression levels in the testes of Miwi catalytic mutant mice. Reporter-based assays further revealed the importance of base pairing between piRNAs and mRNA targets and the requirement for both the slicer activity and piRNA-loading ability of MIWI in piRNA-mediated target repression. Importantly, we demonstrated that proper turnover of certain key piRNA targets is essential for sperm formation. Together, these findings reveal the siRNA-like function of the piRNA machinery in mouse testes and its central requirement for male germ cell development and maturation.

Spatial and temporal translational control of germ cell mRNAs mediated by the eIF4E isoform IFE-1.

Regulated mRNA translation is vital for germ cells to produce new proteins in the spatial and temporal patterns that drive gamete development. Translational control involves the de-repression of stored mRNAs and their recruitment by eukaryotic initiation factors (eIFs) to ribosomes. C. elegans expresses five eIF4Es (IFE-1-IFE-5); several have been shown to selectively recruit unique pools of mRNA. Individual IFE knockouts yield unique phenotypes due to inefficient translation of certain mRNAs. Here, we identified mRNAs preferentially translated through the germline-specific eIF4E isoform IFE-1. Differential polysome microarray analysis identified 77 mRNAs recruited by IFE-1. Among the IFE-1-dependent mRNAs are several required for late germ cell differentiation and maturation. Polysome association of gld-1, vab-1, vpr-1, rab-7 and rnp-3 mRNAs relies on IFE-1. Live animal imaging showed IFE-1-dependent selectivity in spatial and temporal translation of germline mRNAs. Altered MAPK activation in oocytes suggests dual roles for IFE-1, both promoting and suppressing oocyte maturation at different stages. This single eIF4E isoform exerts positive, selective translational control during germ cell differentiation.

Is a CIS phenotype apparent in children with Disorders of Sex Development? Milder testicular dysgenesis is associated with a higher risk of malignancy.

All malignant testicular germ cell tumors (TGCT) of adult men are preceded by an in situ stage (CIS) of protracted evolution. The adult CIS is well characterized, but there is debate on the phenotype of infantile CIS, its distinction from delayed maturation of germ cells and prognostic potential. A large series of 43 patients with Disorders of Sex Development (DSD) and dysgenetic testes (90% ranging from neonates to 12years, mean age 4.7years), was studied by quantifying dysgenetic features, degree of germ cell abnormalities/atypia (GCA), expression of OCT 3/4 (a pluripotency-undifferentiation marker), germ cell ploidy and evolution to CIS and invasive TGCT. Findings were compared with those of normal testes. The type of gonads present defined three groups of patients: bilateral testes (BT-DSD, n=21), one testis and one streak gonad (CT-DSD, C for combined, n=13), and ovarian-testicular combinations (OT-DSD, n=9). There were 5 boys with infantile CIS, bilateral in 3 (total of 8 infantile CIS) and two patients with adult CIS, bilateral in one (total of 3 adult CIS). Two patients had bilateral seminomas one at 12-17 and the other at 23years. Histological dysgenesis was significantly higher in CT-DSD (p<0.05), that had only 1 CIS. The highest frequency of GCA was in BT-DSD (p<0.05), which coincided with a total of 11CIS+Seminomas. In all patients, aneuploidy was significantly higher (63%) than diploidy (p<0.02), and GCA were more frequent in aneuploid than in diploid samples (p<0.02). All CIS and TGCT were OCT 3/4 positive. Finally, there was a significant association between the triad Aneuploidy+GCA+OCT 3/4 positivity and the incidence of CIS (Fisher Exact test p<0.002, relative risk 7.0). The degree of testicular dysgenesis (derived from abnormal organization of Sertoli cells in fetal testicular cords) is inversely related to the incidence of CIS. Our data demonstrate that the combined use of OCT 3/4 expression, quantification of germ cell abnormalities-atypia and ploidy in dysgenetic testes can satisfactorily identify infantile CIS with high risk of malignant evolution and set it aside from delayed germ cell maturation with lower or nil neoplastic potential.

Primary Testicular Failure: An Overview

Primary testicular failure (PTF) or hypergonadotropic hypogonadism refers to conditions where testes fail to produce sperm despite adequate hormonal support. Primary testicular failure is major cause of non-obstructive azoospermia and oligospermia. It affects approximately 1% of all men and 10% of those seeking fertility evaluations [1]. Non-obstructive azoospermia i.e. defect in spermatogenesis can be primary (testicular pathology) or secondary (pathology outside testes; mostly hypothalamo-pituitary axis or pretesticular). The pre-testicular (hypogonadotropic hypogonadism) causes of azoospermia may be defined as extragonadal endocrine disorder originating in the pineal, hypothalamus, pituitary, or adrenals, which have an adverse effect on spermatogenesis through aberrant hormonal action. The testicular (hypergonadotropic hypogonadism/ primary testicular failure) causes of azoospermia are primary defects of the testes. Primary testicular failure is classified into four distinct subtypes viz., sertoli cell only syndrome (SCOS), germ cell/maturation arrest (GCA/MA), hypospermatogenesis (HS) and tubular fibrosis (TF) according to histopathology/cytology findings although accurate categorization is only possible by multiple testicular biopsies/fine needle aspiration cytology (FNAC). However, often one may find differences in subtypes between testes viz., SCOS in one side and tubular fibrosis in other side or any other combinations and these cases are usually labeled as mixed group. In addition many patients may present as complete germinal cell aplasia in some tubules whereas complete spermatogenesis in adjacent tubules (focal germinal cell aplasia) or some tubules with sertoli cells only/hyaline sclerosis and other tubules with complete spermatogenesis or any combinations. This categorization should be considered as a description of histopathologic phenotypes of spermatogenic failure, and not as manifestations of disease entities. Sometimes, patients may present as hypospermatogenesis/maturation arrest initially and later as sertoli cell only syndrome/testicular fibrosis over a period of few years hence diagnosis may change with time. Sertoli cell only syndrome or germ cell aplasia applies to a testis, in which germ cells at any stage are absent, but the tubular architecture is not affected by fibrosis, and supporting cells continue to be present. Sertoli cells play a central role in development of a functional testis, and hence in the expression of a male phenotype. Sertoli cell only syndrome is a common finding of non-obstructive azoospermia. It is a histopathologic phenotype of spermatogenic failure and described first by Castillo et al. [2], in complete germ cell aplasia, the tubules are reduced in diameter, contain only sertoli cells and no other cells involved in spermatogenesis are present. The primordial germ cells either do not migrate from the yolk sac into the future gonads or do not survive in the seminiferous tubules. Germ cell aplasia can also be focal with a variable percentage of tubules containing germ cells, but even in these tubules, spermatogenesis is often have limited activity [3] and hence, should be categorized as hypospermatogenesis. Maturation/Germ Cell arrest is the interruption of normal germ cell maturation at a specific cell stage including spermatogonia, spermatocyte or spermatid level. In hypospermatogenesis all the stages of spermatogenesis are present in some or all tubules, but they are reduced in number. Tubular fibrosis presents as thickening of peritubular membrane and hyaline deposition on basement membrane with absence of germ cells and sertoli cells.

Processive pulses of retinoic acid propel asynchronous and continuous murine sperm production.

The asynchronous cyclic nature of spermatogenesis is essential for continual sperm production and is one of the hallmarks of mammalian male fertility. While various mRNA and protein localization studies have indirectly implicated changing retinoid levels along testis tubules, no quantitative evidence for these changes across the cycle of the seminiferous epithelium currently exists. This study utilized a unique mouse model of induced synchronous spermatogenesis, localization of the retinoid-signaling marker STRA8, and sensitive quantification of retinoic acid concentrations to determine whether there are fluctuations in retinoid levels at each of the individual stages of germ cell differentiation and maturation to sperm. These data show that processive pulses of retinoic acid are generated during spermatogonial differentiation and are the likely trigger for cyclic spermatogenesis and allow us, for the first time, to understand how the cycle of the seminiferous epithelium is generated and maintained. In addition, this study represents the first direct quantification of a retinoid gradient controlling cellular differentiation in a postnatal tissue.