Germ Cell Arrest Research Articles

MicroRNAs in Sertoli cells: implications for spermatogenesis and fertility.

In recent decades, infertility has been considered a major widespread public health issue of very high concern. Currently, almost 50% of infertility cases are due to male factors, including semen disorders, obstructions, cryptorchidism, varicocele and testicular failures, which can occur due to malfunctions in both somatic and germ cells. In this context, besides other approaches, different miRNAs have been used as biomarkers for the diagnosis of male infertility, with different pathologic conditions such as Sertoli cell-only syndrome, mixed atrophy, and germ cell arrest. However, most studies related to male fertility do not point out the functions and cell targets of the described miRNAs. Initial investigations using experimental assays in murine and porcine models were performed, providing the first evidence of the influence of miRNAs on Sertoli cell function including, for instance, proliferation, maturation and hormone responses of these cells. The aim of this mini-review is therefore to summarize our present knowledge of this relevant subject and to highlight the importance of future investigations concerning the miRNA influence in the control of Sertoli cells, spermatogenesis and male fertility.

DMRT proteins and coordination of mammalian spermatogenesis

DMRT genes encode a deeply conserved family of transcription factors that share a unique DNA binding motif, the DM domain. DMRTs regulate development in a broad variety of metazoans and they appear to have controlled sexual differentiation for hundreds of millions of years. In mice, starting during embryonic development, three Dmrt genes act sequentially to help establish and maintain spermatogenesis. Dmrt1 has notably diverse functions that include repressing pluripotency genes and promoting mitotic arrest in embryonic germ cells, reactivating prospermatogonia perinatally, establishing and maintaining spermatogonial stem cells (SSCs), promoting spermatogonial differentiation, and controlling the mitosis/meiosis switch. Dmrt6 acts in differentiating spermatogonia to coordinate an orderly exit from the mitotic/spermatogonial program and allow proper timing of entry to the meiotic/spermatocyte program. Finally, Dmrt7 takes over during the first meiotic prophase to help choreograph a transition in histone modifications that maintains transcriptional silencing of the sex chromosomes. The combined action of these three Dmrt genes helps ensure robust and sustainable spermatogenesis.

Speedy A–Cdk2 binding mediates initial telomere–nuclear envelope attachment during meiotic prophase I independent of Cdk2 activation

Telomere attachment to the nuclear envelope (NE) is a prerequisite for chromosome movement during meiotic prophase I that is required for pairing of homologous chromosomes, synapsis, and homologous recombination. Here we show that Speedy A, a noncanonical activator of cyclin-dependent kinases (Cdks), is specifically localized to telomeres in prophase I male and female germ cells in mice, and plays an essential role in the telomere-NE attachment. Deletion of Spdya in mice disrupts telomere-NE attachment, and this impairs homologous pairing and synapsis and leads to zygotene arrest in male and female germ cells. In addition, we have identified a telomere localization domain on Speedy A covering the distal N terminus and the Cdk2-binding Ringo domain, and this domain is essential for the localization of Speedy A to telomeres. Furthermore, we found that the binding of Cdk2 to Speedy A is indispensable for Cdk2's localization on telomeres, suggesting that Speedy A and Cdk2 might be the initial components that are recruited to the NE for forming the meiotic telomere complex. However, Speedy A-Cdk2-mediated telomere-NE attachment is independent of Cdk2 activation. Our results thus indicate that Speedy A and Cdk2 might mediate the initial telomere-NE attachment for the efficient assembly of the telomere complex that is essential for meiotic prophase I progression.

Histological, immunohistochemical, and morphometric study of peritubular cells and mast cells in the testicular interstitium of azoospermic men

Background Infertility continues to be a highly prevalent condition. Although assisted reproduction technology has been significantly effective, it is relatively costly and may yield disappointing clinical outcomes. There is still an unexplained correlation between the substantial contribution of the testicular somatic cells such as the peritubular cells and interstitial immune cells such as mast cells. Aim The aim of this work was to study the histological, immunohistochemical, and morphometric profiles of peritubular cells and mast cells in azoospermic men that might be a clue for male infertility therapy. Patients and methods This study was carried out on 39 infertile azoospermic men attending the Andrology Outpatient Clinic in Assiut University Hospital. Patients were divided into two groups. Group I (GI), which included patients with obstructive azoospermia (n=9), and group II (GII), which included patients with nonobstructive azoospermia. GII was subdivided as follows: subgroup A, which included patients with Sertoli cell-only syndrome (n=9); subgroup B, which included patients with germ cell arrest (n=10); and subgroup C, which included patients with hypospermatogenesis (n=11). Testicular biopsies were obtained and processed for light, immunohistochemical, morphometric analysis, and electron microscopic examinations. Results The testicular interstitium of GI showed an apparently normal pattern of organization as regards cellular and fibrous components. GIIA showed the highest mast cell number, which established cytoplasmic projections toward peritubular cells. The mean count of tumor necrosis factor α-positive cells showed a significant increase in GIIA compared with GI. The mean area% of collagen fibers increased significantly in GIIA, GIIB, and GIIC compared with GI. In GIIB, bundles of microfibrils were most evident between the basement membrane and the first layer of peritubular cells. In GIIC, they appeared flattened with slender interlacing processes forming bridges. Mast cells bore long cytoplasmic projections, which extended toward the neighboring fibroblasts. Conclusion The present data signify complex cell interactions in cases of testicular dysfunction within the interstitial compartment.

Inhibitory phosphorylation of Cdk1 mediates prolonged prophase I arrest in female germ cells and is essential for female reproductive lifespan.

A unique feature of female germ cell development in mammals is their remarkably long arrest at the prophase of meiosis I, which lasts up to 50 years in humans. Both dormant and growing oocytes are arrested at prophase I and completely lack the ability to resume meiosis. Here, we show that the prolonged meiotic arrest of female germ cells is largely achieved via the inhibitory phosphorylation of Cdk1 (cyclin-dependent kinase 1). In two mouse models where we have introduced mutant Cdk1T14AY15F which cannot be inhibited by phosphorylation (Cdk1AF) in small meiotically incompetent oocytes, the prophase I arrest is interrupted, leading to a premature loss of female germ cells. We show that in growing oocytes, Cdk1AF leads to premature resumption of meiosis with condensed chromosomes and germinal vesicle breakdown followed by oocyte death, whereas in dormant oocytes, Cdk1AF leads to oocyte death directly, and both situations damage the ovarian reserve that maintains the female reproductive lifespan, which should be around 1 year in mice. Furthermore, interruption of the inhibitory phosphorylation of Cdk1 results in DNA damage, which is accompanied by induction of the Chk2 (checkpoint kinase 2)-p53/p63-dependent cell death pathway, which eventually causes global oocyte death. Together, our data demonstrate that the phosphorylation-mediated suppression of Cdk1 activity is one of the crucial factors that maintain the lengthy prophase arrest in mammalian female germ cells, which is essential for preserving the germ cell pool and reproductive lifespan in female mammals.

Relationship between AZFc deletions and testicular histology in infertile South Chinese men with azoospermia and severe oligospermia.

BackgroundThe AZFc deletion has been associated with wide range of phenotypes including complete absence of germ cells in the testes (SCOS), reduction in germ cells hypospermatogenesis, and maturation arrest. The main objective of this study was to evaluate the relationship between AZFc microdeletions and testicular histology in South Chinese men with azoospermia or severe oligospermia.Findings338 men presenting with idiopathic non-obstructive azoospermia or severe oligospermia were evaluated between March 2012 and April 2015. Thirty-nine of the patients examined had an AZFc deletion (10.9 %). Testicular cytopathology was examined in 25 patients with an AZFc microdeletion and 14 with an AZFc deletion. There was no significant difference in the testicular histology of patients with partial or complete AZFc deletions (Mann–Whitney U = 152.500, p = 0.515). There was an association between testicular histology and gr/gr, b1/b3 or b2/b3 deletion (Fisher’s exact test, p = 0.013).ConclusionsMen with a gr/gr partial deletion were at higher risk of having hypospermatogenesis or maturation arrest. Men with a b1/b3 partial deletion were at higher risk of having maturation arrest. Men with a b2/b3 partial deletion were at higher risk of having maturation arrest or complete absence of germ cells in the testes.

Radiofrequency radiation (900 MHz)-induced DNA damage and cell cycle arrest in testicular germ cells in swiss albino mice.

Even though there are contradictory reports regarding the cellular and molecular changes induced by mobile phone emitted radiofrequency radiation (RFR), the possibility of any biological effect cannot be ruled out. In view of a widespread and extensive use of mobile phones, this study evaluates alterations in male germ cell transformation kinetics following RFR exposure and after recovery. Swiss albino mice were exposed to RFR (900 MHz) for 4 h and 8 h duration per day for 35 days. One group of animals was terminated after the exposure period, while others were kept for an additional 35 days post-exposure. RFR exposure caused depolarization of mitochondrial membranes resulting in destabilized cellular redox homeostasis. Statistically significant increases in the damage index in germ cells and sperm head defects were noted in RFR-exposed animals. Flow cytometric estimation of germ cell subtypes in mice testis revealed 2.5-fold increases in spermatogonial populations with significant decreases in spermatids. Almost fourfold reduction in spermatogonia to spermatid turnover (1C:2C) and three times reduction in primary spermatocyte to spermatid turnover (1C:4C) was found indicating arrest in the premeiotic stage of spermatogenesis, which resulted in loss of post-meiotic germ cells apparent from testis histology and low sperm count in RFR-exposed animals. Histological alterations such as sloughing of immature germ cells into the seminiferous tubule lumen, epithelium depletion and maturation arrest were also observed. However, all these changes showed recovery to varied degrees following the post-exposure period indicating that the adverse effects of RFR on mice germ cells are detrimental but reversible. To conclude, RFR exposure-induced oxidative stress causes DNA damage in germ cells, which alters cell cycle progression leading to low sperm count in mice.

Dynamin 2 is essential for mammalian spermatogenesis.

The dynamin family of proteins play important regulatory roles in membrane remodelling and endocytosis, especially within brain and neuronal tissues. In the context of reproduction, dynamin 1 (DNM1) and dynamin 2 (DNM2) have recently been shown to act as key mediators of sperm acrosome formation and function. However, little is known about the roles that these proteins play in the developing testicular germ cells. In this study, we employed a DNM2 germ cell-specific knockout model to investigate the role of DNM2 in spermatogenesis. We demonstrate that ablation of DNM2 in early spermatogenesis results in germ cell arrest during prophase I of meiosis, subsequent loss of all post-meiotic germ cells and concomitant sterility. These effects become exacerbated with age, and ultimately result in the demise of the spermatogonial stem cells and a Sertoli cell only phenotype. We also demonstrate that DNM2 activity may be temporally regulated by phosphorylation of DNM2 via the kinase CDK1 in spermatogonia, and dephosphorylation by phosphatase PPP3CA during meiotic and post-meiotic spermatogenesis.

Histological alterations in Leydig cells and macrophages in azoospermic men.

The study aimed to compare the histological features of Leydig cells and macrophages in the testicular interstitium of obstructive versus nonobstructive azoospermia. Thirty-nine azoospermic men undergoing testicular sperm extraction during intracytoplasmic sperm injection were allocated into obstructive azoospermia group (GI) and nonobstructive azoospermia group (GII) which was subdivided into Sertoli cell-only syndrome (GIIA), germ cell arrest (GIIB) and hypospermatogenesis (GIIC) subgroups. Serum LH, FSH and testosterone levels were measured. Ultrastructural changes and the mean number of CD68-positive cells were estimated in the different groups. In GIIA, Leydig cells' processes came in contact with macrophages and showed smooth endoplasmic reticulum dilatation. In GIIB, Leydig cells showed apoptotic changes. Macrophages were commonly encountered in their vicinity demonstrating large number of lysosomes. In GIIC, Leydig cells showed euchromatic nuclei. Macrophages showed expulsion of their lysosomal contents in the interstitium surrounded by apoptotic bodies. The mean count of total CD68-positive macrophages was higher in cases of obstructive azoospermia with nonsignificant differences compared to nonobstructive azoospermia groups. Significant increase in FSH level was detected in GIIA compared to GI. It is concluded that structural interactions might take place between Leydig cells and macrophages in the interstitial tissue of azoospermic men.

EFFECTS OF SIBUTRAMINE AND GREEN COFFEE ON THE HISTOLOGICAL AND ULTRASTRUCTURAL ALTERATIONS IN THE TESTES OF RATS INDUCED BY OBESITY

Obesity is a multi-factorial chronic disorder which causes some serious problems. Sibutramine is anti-obesity drug with established efficacy in sustained weight reduction, but it has a side effect on blood pressure and heart rate elevation. The present study aimd to pinpointing the effects of sibutramine and green coffee in ameliorating the histological and ultrastructural disorders in the testes of rats induced by obesity. This study was assessed in male albino rats (120-150 g). The rats were divided into four groups: the 1st group received a normal diet and acted as a control negative, the 2nd high fat diet (HFD) group acted as control positive, the 3rd HFD group treated with sibutramine (10 mg/kg body weight/day), and the 4th HFD group treated with green coffee (100 mg/kg body weight/day). The present histological study revealed that sibutramine caused histopathological changes in the testes of obese rat including vacuolation, maturation arrest and exfoliation of germ cells in the lumina of the seminiferous tubules. Moreover, congestion of the intertubular blood vessels and proliferation of Leydig cells in the interstitial tissue were also observed. Ultrastructurally, the boundary tissue of some tubules showed marked changes; it was folded and thickened. Sertoli cells showed cytoplasmic vacuolation, vacuolated mitochondria, increased number of lysosomes and their nuclei showed signs of pyknosis. Moreover, Leydig cells revealed vacuolation and decrease in the number of lipid droplets. On the other hand, treatment of the obese rats with green coffee induced great recovery of the damaged testicular tissues. This improvement may be due to the antioxidant properties and scavenging abilities of green coffee against active free radicals.

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.

Prostaglandin D2 acts through the Dp2 receptor to influence male germ cell differentiation in the foetal mouse testis.

Through intercellular signalling, the somatic compartment of the foetal testis is able to program primordial germ cells to undergo spermatogenesis. Fibroblast growth factor 9 and several members of the transforming growth factor β superfamily are involved in this process in the foetal testis, counteracting the induction of meiosis by retinoic acid and activating germinal mitotic arrest. Here, using in vitro and in vivo approaches, we show that prostaglandin D2 (PGD2), which is produced through both L-Pgds and H-Pgds enzymatic activities in the somatic and germ cell compartments of the foetal testis, plays a role in mitotic arrest in male germ cells by activating the expression and nuclear localization of the CDK inhibitor p21(Cip1) and by repressing pluripotency markers. We show that PGD2 acts through its Dp2 receptor, at least in part through direct effects in germ cells, and contributes to the proper differentiation of male germ cells through the upregulation of the master gene Nanos2. Our data identify PGD2 signalling as an early pathway that acts in both paracrine and autocrine manners, and contributes to the differentiation of germ cells in the foetal testis.

Meiosis-Specific Cohesin Component, Stag3 Is Essential for Maintaining Centromere Chromatid Cohesion, and Required for DNA Repair and Synapsis between Homologous Chromosomes

Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin complexes. Furthermore, our data suggests that STAG3 is required for structural changes of chromosomes that mediate chromosome pairing and synapsis, DNA repair and progression of meiosis.

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

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

Doxycyclin ameliorates a starvation-induced germline tumor in C. elegans daf-18/PTEN mutant background

Managing available resources is a key necessity of each organism to cope with the environment. The nematode C. elegans responds to nutritional deprivation or harsh environmental conditions with a multitude of developmental adaptations, among them a starvation-induced quiescence at early larval development (L1). daf-18, the C. elegans homolog of the human tumor suppressor gene PTEN, is essential for the maintenance of survival and germline stem cell arrest during the L1 diapause. We show here that daf-18 mutants, independently to their failure to maintain G2 arrest of the primordial germ cells, develop a gonad phenotype after refeeding. This highly penetrant gonadal phenotype is further enhanced by a mutation in shc-1, encoding a protein homologous to the human adaptor ShcA. Features of this phenotype are a tumor-like phenotype encompassing hyper-proliferation of germ cell nuclei and disruption/invasion of the basement membrane surrounding the gonad. The penetrance of this phenotype is reduced by decreasing starvation temperature. In addition, it is also ameliorated in a dose-dependent way by exposure to the antibiotic doxycyclin either during starvation or during subsequent refeeding. Since, in eukaryotic cells, doxycyclin specifically blocks mitochondrial translation, our results suggest that daf-18 and shc-1;daf-18 mutants fail to adapt mitochondrial activity to reduced nutritional availability during early larval developing.

Evaluation on Changes of Testicular Histology in Aging Men

Objective To evaluate the changes of testicular histology in aging men. Methods Testicular tissues were obtained by orchiectomy from 40 aging men (75.9 ± 3.8 years) with advanced prostate cancers (aging group). Control group comprised 20 testicular tissue samples obtained by biopsy from young men with obstructive azoospermia (33.7 ± 4.9 years) who underwent assisted reproductive treatment. Hematoxylin and eosin were used to stain the sections for observation of histology. Changes of seminiferous tubules and interstitial compartment were observed. Results In aging group, vacuolation of seminiferous epithelium was a common phenomenon and the incidence was 85%. Ten samples had moderate spermatogenesis. Thirty cases showed hypospermatogenesis or germ cell arrest, even absence of spermatogenesis. In samples with germ cell arrest, seminiferous epithelia had only the layer of spermatogonia. Twenty-three cases had the heterogeneity of spermatogenesis. In interstitial compartment, pathological changes included loose connection and Leydig cells aggregations and reduce. In the control, most samples demonstrated a normal spermatogenesis. Conclusion Pathological changes occurred in aging testis were associated with degenerative alterations on both the seminiferous epithelium and the interstitial compartment, which contribute to lowering testis functions.

Evidence that active demethylation mechanisms maintain the genome of carcinoma in situ cells hypomethylated in the adult testis

Background:Developmental arrest of fetal germ cells may lead to neoplastic transformation and formation of germ cell tumours via carcinoma in situ (CIS) cells. Normal fetal germ cell development requires complete erasure and re-establishment of DNA methylation. In contrast to normal spermatogonia, the genome of CIS cells remains unmethylated in the adult testis. We here investigated the possible active and passive pathways that can sustain the CIS genome hypomethylated in the adult testis.Methods:The levels of 5-methyl-cytosine (5mC) and 5-hydroxy-methyl-cytosine (5hmC) in DNA from micro-dissected CIS cells were assessed by quantitative measurements. The expression of TET1, TET2, APOBEC1, MBD4, APEX1, PARP1, DNMT1, DNMT3A, DNMT3B and DNMT3L in adult testis specimens with CIS and in human fetal testis was investigated by immunohistochemistry and immunofluorescence.Results:DNA from micro-dissected CIS cells contained very low levels of 5hmC produced by ten eleven translocation (TET) enzymes. CIS cells and fetal germ cells expressed the suggested initiator of active demethylation, APOBEC1, and the base excision repair proteins MBD4, APEX1 and PARP1, whereas TETs – the alternative initiators were absent. Both maintenance and de novo methyltransferases were detected in CIS cells.Conclusion:The data are consistent with the presence of an active DNA de-methylation pathway in CIS cells. The hypomethylated genome of CIS cells may contribute to phenotypic plasticity and invasive capabilities of this testicular cancer precursor.

MicroRNA expression profiles in human testicular tissues of infertile men with different histopathologic patterns

To investigate the expression profiles of microRNA (miRNA) in human testes showing different histopathological patterns. Microarray with quantitative real-time polymerase chain reaction (qRT-PCR) validation. University research and clinical institutes. Azoospermic men who underwent testicular biopsy for sperm recovery in preparation for intracytoplasmic sperm injection. Testicular biopsies. Statistically significantly altered miRNA expression profiles among the testicular histopathologic patterns groups compared with normal pattern group. According to miRNA array, a total of 197, 68, and 46 miRNAs were found to be differentially expressed when comparing the samples from Sertoli cell only (SCO), mixed atrophy (MA), and germ cell arrest (GA) groups, respectively, with normal spermatogenesis (N). Five miRNAs have been validated using qRT-PCR, and the results were consistent with miRNA array analysis. Bioinformatics analysis showed that five microRNAs (hsa-mir-34b*, hsa-mir-34b, hsa-mir-34c-5p, hsa-mir-449a, and hsa-mir-449b*) were involved in apoptosis, cell proliferation, and differentiation. Notably, potential target genes of these five miRNAs were involved in the spermatogenesis process. This study provides new insights into specific miRNAs that are expressed in infertile men with different histopathologic patterns, suggesting a role of miRNAs in regulating male germ and somatic cells and that their alteration is associated with reproductive abnormalities.

A retrospective analysis of pathological changes of testicular tissue in normal adult rats

Rat testicular model is widely used in experiments on andrology, pharmacology and reproductive toxicology. Generally, normal adult rat is considered to have normal testes. However, whether normal adult rats appeared abnormal testes have not been evaluated. The objective of this study was to evaluate the incidence of abnormal testes in normal adult Sprague Dawley (SD) rats and pathological changes in testicular tissues. Six hundred and sixteen adult male SD rats used in previous studies as controls were retrospectively analysed. Testicular tissues were stained with haematoxylin-eosin for observation of pathology. Among 616 rats, 14 rats had pathological testes, and the incidence of abnormal testis was 2.3%. In the 14 rats with abnormal testes, 10 rats were microrchidia (71.4%) and four rats showed normal testicular size. Testicular abnormality included complete interruption of spermatogenesis, partial germ cell arrest, progressive hypospermatogenesis, seminiferous epithelia vacuolation and inflammatory status. Bilateral testicular tissues had similar pathological changes in abnormal testes. The findings in this study demonstrate that the normal adult rats have abnormal testes. We should pay attention to the possibility of abnormal testes when using normal adult male rats for establishing a testicular model.

Postnatal epigenetic reprogramming in the germline of a marsupial, the tammar wallaby

BackgroundEpigenetic reprogramming is essential to restore totipotency and to reset genomic imprints during mammalian germ cell development and gamete formation. The dynamic DNA methylation change at DMRs (differentially methylated regions) within imprinted domains and of retrotransposons is characteristic of this process. Both marsupials and eutherian mammals have genomic imprinting but these two subgroups have been evolving separately for up to 160 million years. Marsupials have a unique reproductive strategy and deliver tiny, altricial young that complete their development within their mother's pouch. Germ cell proliferation in the genital ridge continues after birth in the tammar wallaby (Macropus eugenii), and it is only after 25 days postpartum that female germ cells begin to enter meiosis and male germ cells begin to enter mitotic arrest. At least two marsupial imprinted loci (PEG10 and H19) also have DMRs. To investigate the evolution of epigenetic reprogramming in the marsupial germline, here we collected germ cells from male pouch young of the tammar wallaby and analysed the methylation status of PEG10 and H19 DMR, an LTR (long terminal repeat) and a non-LTR retrotransposons.ResultsDemethylation of the H19 DMR was almost completed by 14 days postpartum and de-novo methylation started from 34 days postpartum. These stages correspond to 14 days after the completion of primordial germ cell migration into genital ridge (demethylation) and 9 days after the first detection of mitotic arrest (re-methylation) in the male germ cells. Interestingly, the PEG10 DMR was already unmethylated at 7 days postpartum, suggesting that the timing of epigenetic reprogramming is not the same at all genomic loci. Retrotransposon methylation was not completely removed after the demethylation event in the germ cells, similar to the situation in the mouse.ConclusionsThus, despite the postnatal occurrence of epigenetic reprogramming and the persistence of genome-wide undermethylation for 20 days in the postnatal tammar, the relative timing and mechanism of germ cell reprogramming are conserved between marsupials and eutherians. We suggest that the basic mechanism of epigenetic reprogramming had already been established before the marsupial-eutherian split and has been faithfully maintained for at least 160 million years and may reflect the timing of the onset of mitotic arrest in the male germline.