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.

As daughter centrioles assemble during G2, they recruit conserved Ana3/RTTN followed by its partner Rcd4/PPP1R35. Together, this contributes to the subsequent recruitment of Ana1/CEP295, required for the centriole\'s conversion to a centrosome. Here, we show that Rcd4/PPP1R35 is also required to maintain 9-fold centriole symmetry in the Drosophila male germline; its absence causes microtubule triplets to disperse into a reduced number of doublet or singlet microtubules. rcd4-null mutant spermatocytes display skinny centrioles that elongate normally and localize centriolar components correctly. Mutant spermatocytes also have centrioles of normal girth that splay at their proximal ends when induced to elongate by Ana1 overexpression. Skinny and splayed spermatid centrioles can still recruit a proximal centriole-like (PCL) structure marking a capability to initiate features of centriole duplication in developing sperm. Thus, stable 9-fold symmetry of microtubule triplets is not essential for centriole growth, correct longitudinal association of centriole components, and aspects of centriole duplication.

Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.

Calcium binding protein, spermatid associated 1 (CABS1) is a protein most widely studied in spermatogenesis. However, mRNA for CABS1 has been found in numerous tissues, albeit with little information about the protein. Previously, we identified CABS1 mRNA and protein in human salivary glands and provided evidence that in humans CABS1 contains a heptapeptide near its carboxyl terminus that has anti-inflammatory activities. Moreover, levels of an immunoreactive form of CABS1 were elevated in psychological stress. To more fully characterize human CABS1 we developed additional polyclonal and monoclonal antibodies to different sections of the protein and used these antibodies to characterize CABS1 in an overexpression cell lysate, human salivary glands, saliva, serum and testes using western blot, immunohistochemistry and bioinformatics approaches exploiting the Gene Expression Omnibus (GEO) database. CABS1 appears to have multiple molecular weight forms, consistent with its recognition as a structurally disordered protein, a protein with structural plasticity. Interestingly, in human testes, its cellular distribution differs from that in rodents and pigs, and includes Leydig cells, primary spermatogonia, Sertoli cells and developing spermatocytes and spermatids, Geodata suggests that CABS1 is much more widely distributed than previously recognized, including in the urogenital, gastrointestinal and respiratory tracts, as well as in the nervous system, immune system and other tissues. Much remains to be learned about this intriguing protein.

The tripartite motif-containing protein 66 (TRIM66, also known as TIF1-delta) is a PHD-Bromo-containing protein primarily expressed in post-meiotic male germ cells known as spermatids. Biophysical assays showed that the TRIM66 PHD-Bromodomain binds to H3 N-terminus only when lysine 4 is unmethylated. We addressed TRIM66\'s role in reproduction by loss-of-function genetics in the mouse. Males homozygous for Trim66-null mutations produced functional spermatozoa. Round spermatids lacking TRIM66 up-regulated a network of genes involved in histone acetylation and H3K4 methylation. Profiling of H3K4me3 patterns in the sperm produced by the Trim66-null mutant showed minor alterations below statistical significance. Unexpectedly, Trim66-null males, but not females, sired pups overweight at birth, hence revealing that Trim66 mutations cause a paternal effect phenotype.

Unlike coding genes, the number of lncRNA genes in organism genomes is relatively proportional to organism complexity. From plants to humans, the tissues with highest numbers and levels of lncRNA gene expression are the male reproductive organs. To learn why, we initiated a genome-wide analysis of Drosophila lncRNA spatial expression patterns in these tissues. The numbers of genes and levels of expression observed greatly exceed those previously reported, due largely to a preponderance of non-polyadenylated transcripts. In stark contrast to coding genes, the highest numbers of lncRNAs expressed are in post-meiotic spermatids. Correlations between expression levels, localization and previously performed genetic analyses indicate high levels of function and requirement. More focused analyses indicate that lncRNAs play major roles in evolution by controlling transposable element activities, Y chromosome gene expression and sperm construction. A new type of lncRNA-based particle found in seminal fluid may also contribute to reproductive outcomes.

CRISPR-Cas9 short guide RNA (sgRNA) library screening is a powerful approach to understand the molecular mechanisms of biological phenomena. However, its in vivo application is currently limited. Here, we developed our previously established in vitro revival screening method into an in vivo one to identify factors involved in spermatogenesis integrity by utilizing sperm capacitation as an indicator. By introducing an sgRNA library into testicular cells, we successfully pinpointed the retinal degeneration 3 (Rd3) gene as a significant factor in spermatogenesis. Single-cell RNA sequencing (scRNA-seq) analysis highlighted the high expression of Rd3 in round spermatids, and proteomics analysis indicated that Rd3 interacts with mitochondria. To search for cell-type-specific signaling pathways based on scRNA-seq and proteomics analyses, we developed a computational tool, Hub-Explorer. Through this, we discovered that Rd3 modulates oxidative stress by regulating mitochondrial distribution upon ciliogenesis induction. Collectively, our screening system provides a valuable in vivo approach to decipher molecular mechanisms in biological processes.

Transposable elements (TEs) are a major constituent of human genes, occupying approximately half of the intronic space. During pre-messenger RNA synthesis, intronic TEs are transcribed along with their host genes but rarely contribute to the final mRNA product because they are spliced out together with the intron and rapidly degraded. Paradoxically, TEs are an abundant source of RNA-processing signals through which they can create new introns1, and also functional2 or non-functional chimeric transcripts3. The rarity of these events implies the existence of a resilient splicing code that is able to suppress TE exonization without compromising host pre-mRNA processing. Here we show that SAFB proteins protect genome integrity by preventing retrotransposition of L1 elements while maintaining splicing integrity, via prevention of the exonization of previously integrated TEs. This unique dual role is possible because of L1\'s conserved adenosine-rich coding sequences that are bound by SAFB proteins. The suppressive activity of SAFB extends to tissue-specific, giant protein-coding cassette exons, nested genes and Tigger DNA transposons. Moreover, SAFB also suppresses LTR/ERV elements in species in which they are still active, such as mice and flies. A significant subset of splicing events suppressed by SAFB in somatic cells are activated in the testis, coinciding with low SAFB expression in postmeiotic spermatids. Reminiscent of the division of labour between innate and adaptive immune systems that fight external pathogens, our results uncover SAFB proteins as an RNA-based, pattern-guided, non-adaptive defence system against TEs in the soma, complementing the RNA-based, adaptive Piwi-interacting RNA pathway of the germline.

Cis-genetic effects are key determinants of transcriptional divergence in discrete tissues and cell types. However, how cis- and trans-effects act across continuous trajectories of cellular differentiation in vivo is poorly understood. Here, we quantify allele-specific expression during spermatogenic differentiation at single-cell resolution in an F1 hybrid mouse system, allowing for the comprehensive characterisation of cis- and trans-genetic effects, including their dynamics across cellular differentiation. Collectively, almost half of the genes subject to genetic regulation show evidence for dynamic cis-effects that vary during differentiation. Our system also allows us to robustly identify dynamic trans-effects, which are less pervasive than cis-effects. In aggregate, genetic effects were strongest in round spermatids, which parallels their increased transcriptional divergence we identified between species. Our approach provides a comprehensive quantification of the variability of genetic effects in vivo, and demonstrates a widely applicable strategy to dissect the impact of regulatory variants on gene regulation in dynamic systems.

In mammals, the adult testis is the tissue with the highest diversity in gene expression. Much of that diversity is attributed to germ cells, primarily meiotic spermatocytes and postmeiotic haploid spermatids. Exploiting a newly developed cell purification method, we profiled the transcriptomes of such postmitotic germ cells of mice. We used a de novo transcriptome assembly approach and identified thousands of novel expressed transcripts characterized by features distinct from those of known genes. Novel loci tend to be short in length, monoexonic, and lowly expressed. Most novel genes have arisen recently in evolutionary time and possess low coding potential. Nonetheless, we identify several novel protein-coding genes harboring open reading frames that encode proteins containing matches to conserved protein domains. Analysis of mass-spectrometry data from adult mouse testes confirms protein production from several of these novel genes. We also examine overlap between transcripts and repetitive elements. We find that although distinct families of repeats are expressed with differing temporal dynamics during spermatogenesis, we do not observe a general mode of regulation wherein repeats drive expression of nonrepetitive sequences in a cell type-specific manner. Finally, we observe many fairly long antisense transcripts originating from canonical gene promoters, pointing to pervasive bidirectional promoter activity during spermatogenesis that is distinct and more frequent compared with somatic cells.