Search

Background: Spermatogenesis is a temperature-sensitive process, and elevation in temperature hampers this process quickly and significantly. We studied the molecular effects of testicular heating on piRNAs and gene expression in rat testicular germ cells. Methods: We generated a cryptorchid rat model by displacing the testis from the scrotal sac (34 °C) to the abdominal area (37 °C) and sacrificed animals after 1 day, 3 days, and 5 days. Pachytene spermatocytes and round spermatids were purified using elutriation centrifugation and percoll gradient methods. We performed transcriptome sequencing in pachytene spermatocytes and round spermatids to identify differentially expressed piRNAs and their probable targets, i.e., TE transcripts and mRNAs. Results: As a result of heat stress, we observed significant upregulation of piRNAs and TE transcripts in testicular germ cells. In addition to this, piRNA biogenesis machinery and heat shock proteins (Hsp70 and Hsp90 family members) were upregulated. mRNAs have also been proposed as targets for piRNAs; therefore, we shortlisted certain piRNA-mRNA pairs with an inverse relationship of expression. We observed that in testicular heat stress, the heat shock proteins go hand-in-hand with the upregulation of piRNA biogenesis machinery. The dysregulation of piRNAs in heat-stressed germ cells, increased ping-pong activity, and disturbed expression of piRNA target transcripts suggest a connection between piRNAs, mRNAs, and TE transcripts. Conclusions: In heat stress, piRNAs, piRNA machinery, and heat shock proteins are activated to deal with low levels of stress, which is followed by a rescue approach in prolonged stressaccompained by high TE activity to allow genetic mutations, perhaps for survival and adaptability. [ABSTRACT FROM AUTHOR]

Spermatogenesis is a delicate and complex biological process in which spermatogonial stem cells continue to proliferate and differentiate into mature spermatozoa, maintaining sperm production in male mammals throughout the lifetime. To study the molecular mechanism of spermatogenesis, researchers had to isolate different germ cell subpopulations for in vitro culture and characterization. However, due to the existence of several stages of germ cells and a variety of populations of somatic cells in the testis of male mammals, it is a challenge for us to obtain high-purity germ cell subpopulations for further research. Here, we optimized the STA-PUT device and successfully applied it to isolate and purify spermatogonia populations in piglets, and multiple germ cell populations at different developmental stages in testes of adult mice and boars. This work provides a simple platform for germ cell fractionation to facilitate the molecular mechanistic study of animal spermatogenesis in vitro. • We established an optimized velocity sedimentation STA-PUT device for cell fractionation. • The STA-PUT device was applied to isolate multiple germ cell populations in mice and pigs. • The STA-PUT device can isolate spermatogonia, spermatocytes, and spermatids in a high purity and a sufficient number. [ABSTRACT FROM AUTHOR]

Spermatogenesis, an efficient and complex system in male germline development, requires a series of elaborately regulated genetic events in which diploid spermatogonia differentiate into haploid spermatozoa. N6-methyladenosine (m6A) is an important epigenetic RNA modification that occurs during spermatogenesis. ALKBH5 is an m6A eraser and knocking out Alkbh5 increases the level of total m6A methylation and causes male infertility. In this study, comprehensive analyses of MeRIP-seq and RNA-seq data revealed differences between wild-type (WT) and Alkbh5 knockout (KO) mice. In pachytene spermatocytes (PA), 8,151 m6A peaks associated with 9,959 genes were tested from WT and 10,856 m6A peaks associated with 10,016 genes were tested from KO mice. In the round spermatids (RO), 10,271 m6A peaks associated with 10,109 genes were tested from WT mice and 9,559 m6A peaks associated with 10,138 genes were tested from KO mice. The peaks were mainly concentrated in the coding region and the stop codon of the GGAC motif. In addition, enrichment analysis showed significant m6A methylation genes in related pathways in spermatogenesis. Furthermore, we conducted joint analyses of the m6A methylome and RNA transcription, suggesting an m6A regulatory mechanism of gene expression. Finally, seven differentially expressed mRNAs from RNA-seq data in both PA and RO were verified using qPCR. Overall, our study provides new information on m6A modification changes between WT and KO in PA and RO, and may provide new insights into the molecular mechanisms of m6A modification in germ cell development and spermatogenesis. [ABSTRACT FROM AUTHOR]

Gonadotropin Regulated Testicular Helicase (GRTH/DDX25) is member of the DEAD-box family of RNA helicases present in Leydig and germ cells. GRTH is the only family member regulated by hormones, luteinizing hormone, through androgen action. Male mice with knock-out of the GRTH gene are sterile, lack sperm with arrest at round spermatids. GRTH participates on the nuclear export and transport of specific mRNAs, the structural integrity of Chromatoid Bodies of round spermatids, where mRNAs are processed and stored, and in their transit to polyribosomes, where it may regulate translation of relevant genes. GRTH has a central role in the control of germ cell apoptosis and acts as negative regulator of miRNAs which regulate expression of genes involved in the progress of spermatogenesis. In Leydig cells, GRTH gene transcription is regulated by LH via autocrine actions of androgen/androgen receptor and has regulatory effects in steroidogenesis. In germ cells, androgen actions are indirect via receptors in Sertoli cells. Transgenic mice carrying GRTH 5′ flanking region-GFP permitted to discern regions in the gene which directs its expression upstream, in germ cells, and downstream in Leydig cells, and the androgen-regulated transcription at interstitial (autocrine), and germ cell (paracrine) compartments. Further evidence for paracrine actions of androgen/androgen receptor is their transcriptional induction of Germ Cell Nuclear Factor as requisite up-regulator of GRTH gene transcription in round spermatids, linking androgen action to two relevant germ cell genes essential for the progress of spermatogenesis. A missense mutation of R to H at amino acid 242 of GRTH found in 5.8% of a patient population with azoospermia causes loss of the cytoplasmic phospho-GRTH species with preservation of the non-phospho form in transfected cells. Mice with knock-in of the human mutation, lack sperm due to arrest at round spermatids. This model permits to discern the function of phospho-GRTH. The GRTH phospho-site resides at a Threonine structurally adjacent to the mutant site found in patients. Molecular modeling of this site elucidated the amino acids that form the GRTH/PKA interphase and provide the basis for drug design for use as male contraceptive. [ABSTRACT FROM AUTHOR]

Abstract: Chemotherapeutic drugs can affect DNA in male germ cells, thereby impacting on the integrity of the genome transmitted to offspring. Drug metabolizing enzymes can protect cells from xenobiotic insult. We analyzed the expression pattern of such enzymes in isolated round spermatids from rats exposed to drugs used to treat testicular cancer: bleomycin, etoposide, and cisplatin (BEP). The number of isozymes expressed and the overall relative expression values were highest for the glutathione S-transferases (GSTs). Moreover, BEP treatment significantly increased the expression of 8 GSTs and 3 aldehyde dehydrogenases. Increased expression of GST isozymes was confirmed by qRT-PCR and Western blot analysis. Although Gst genes can be targets for epigenetic modifications, promoter DNA methylation was not affected by BEP treatment. As GSTs are involved in drug resistance mechanisms, we hypothesize that BEP induction of GST expression may lead to the survival of damaged germ cells and the production of abnormal sperm. [Copyright &y& Elsevier]

Abstract: Dicer1, an RNase III endonuclease, is indispensable for the maturation of miRNA and siRNA, which control gene expression through the RNAi pathway. The diverse functions of miRNA involving multiple developmental processes have been elucidated, but the role of Dicer1 in spermatogenesis is just beginning to be revealed. Mice lacking Dicer1 were reported to be embryonic lethal at E7.5. In the present study, mice with a Dicer1 conditional allele were crossed with Vasa-cre transgenic mice to delete Dicer1 as early as the prospermatogonia stage (at E15). At P40, seminiferous tubules of Dicer1 deficient mice showed several aberrant phenotypes. A large number of apoptotic germ cells were detected by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, but several events in meiosis of spermatocytes appeared unaffected. The mutant mice were found to be sterile, likely due to the extensive decrease in number and morphological abnormalities of mature sperm in the epididymis, which, together with the numerous haploid cells in the testis, indicated a severely affected transition from round to functional elongated spermatozoa. Additionally, we found milder phenotypes when Dicer1 was inactivated in later stages of spermatogenesis in Stra8-cre and Pgk2-cre transgenic mice. In conclusion, our findings suggest that the loss of Dicer1 has a continuous and cumulative effect on the process of spermatogenesis and blocks the germ cells in the stage of round spermatids to a large extent, ultimately leading to the generation of abnormal sperm. [Copyright &y& Elsevier]

Spermatids are the earliest male germ cells with haploid set of chromosomes. Spermatid injection was introduced in human assisted reproduction for the treatment of men with non- obstructive azoospermia. Spermatozoa can be recovered in half of patients with nonobstructive azoospermia. The use of spermatids for intracytoplasmic injection (ICSI) has been proposed for cases in which no spermatozoa can be retrieved. However, there are low pregnancy rates following ICSI using round spermatids from men with no elongated spermatids or spermatozoa in their testes. The in vitro culture of immature germ cells has been proposed as a means to improve this poor outcome. Oocyte activation rarely occurs when injected with a spermatid. Therefore, spermatid injection requires use of calcium ionophores for oocyte activation which is otherwise carried out by PLC zeta from mature sperms. This is the only option available for the nonobstructive azoospermic patients to have their own biological child. [ABSTRACT FROM AUTHOR]