Male reproductive aging, or andropause, is associated with gradual age-related changes in testicular properties, sperm production, and erectile function. The testis, which is the primary male reproductive organ, produces sperm and androgens. To understand the transcriptional changes underlying male reproductive aging, we performed transcriptome analysis of aging testes in mice. A total of 31,386 mRNAs and 9387 long non-coding RNAs (lncRNAs) were identified in the mouse testes of diverse age groups (3, 6, 12, and 18 months old) by total RNA sequencing. Of them, 1571 mRNAs and 715 lncRNAs exhibited changes in their levels during testicular aging. Most of these aging-related transcripts exhibited slight and continuous expression changes during aging, whereas some (9.6%) showed larger expression changes. The aging-related transcripts could be classified into diverse expression patterns, in which the transcripts changed mainly at 3–6 months or at 12–18 months. Our subsequent in silico analysis provided insight into the potential features of testicular aging-related mRNAs and lncRNAs. We identified testis-specific aging-related transcripts (121 mRNAs and 25 lncRNAs) by comparison with a known testis-specific transcript profile, and then predicted the potential reproduction-related functions of the mRNAs. By selecting transcripts that are altered only between 3 and 18 months, we identified 46 mRNAs and 34 lncRNAs that are stringently related to the terminal stage of male reproductive aging. Some of these mRNAs were related to hormonal regulation. Finally, our in silico analysis of the 34 aging-related lncRNAs revealed that they co-localized with 19 testis-expressed protein-coding genes, 13 of which are considered to show testis-specific or -predominant expression. These nearby genes could be potential targets of cis-regulation by the aging-related lncRNAs. Collectively, our results identify a number of testicular aging-related mRNAs and lncRNAs in mice and provide a basis for the future investigation of these transcripts in the context of aging-associated testicular dysfunction.

Background: Spermatogenesis, which is the complex and highly regulated process of producing haploid spermatozoa, involves testis-specific transcripts. Recent studies have discovered that long noncoding RNAs (IncRNAs) are novel regulatory molecules that play important roles in various biological processes. However, there has been no report on the comprehensive identification of testis-specific IncRNAs in mice. Results: We performed microarray analysis of transcripts from mouse brain, heart, kidney, liver and testis. We found that testis harbored the highest proportion of tissue-specific IncRNAs (11%; 1607 of 14,256). Testis also harbored the largest number of tissue-specific mRNAs among the examined tissues, but the proportion was lower than that of IncRNAs (7%; 1090 of 16,587). We categorized the testis-specific IncRNAs and found that a large portion corresponded to long intergenic ncRNAs (IincRNAs). Genomic analysis identified 250 protein-coding genes located near (<= 10kb) 194 of the loci encoding testis-specific IincRNAs. Gene ontology (GO) analysis showed that these protein-coding genes were enriched for transcriptional regulation-related terms. Analysis of male germ cell-related cell lines (F9, GC-1 and GC-2) revealed that some of the testis-specific IncRNAs were expressed in each of these cell lines. Finally, we arbitrarily selected 26 testis-specific IncRNAs and performed in vitro expression analysis. Our results revealed that all of them were expressed exclusively in the testis, and 23 of the 26 showed germ cell-specific expression. Conclusion: This study provides a catalog of testis-specific IncRNAs and a basis for future investigation of the IncRNAs involved in spermatogenesis and testicular functions.

Heat shock factor 2 (HSF2) regulates the transcription of the male-specific region of the mouse Y chromosome long arm (MSYq) multicopy genes only in testes, but the molecular mechanism underlying this tissue specificity remains largely unknown. Here, we report that the testicular germ cell–specific long noncoding RNA (lncRNA), NR_038002, displays a characteristic spatiotemporal expression pattern in the nuclei of round and elongating spermatids. NR_038002-knockout male mice produced sperm with abnormal head morphology and exhibited reduced fertility accompanied by a female-biased sex ratio in offspring. Molecular analyses revealed that NR_038002 interacts with HSF2 and thereby activates expression of the MSYq genes. We designate NR_038002 as testicular germ cell–specific HSF2-interacting lncRNA (Teshl). Together, our study is the first to demonstrate that the testis specificity of HSF2 activity is regulated by the lncRNA Teshl and establishes a Teshl-HSF2-MSYq molecular axis for normal Y-bearing sperm qualities and consequent balanced offspring sex ratio.

Zfp819, a member of the Krüppel-associated box (KRAB) family, encodes a spermatogenic cell-specific transcription factor. Zfp819-overexpression induces apoptosis and inhibits proliferation in somatic cell lines. In the present study, we examined the cellular effects of Zfp819 in a male germ cell line (GC-2 cells). Overexpression of Zfp819 demonstrated an increase in the number of apoptotic cells, leading to inhibition of proliferation in GC-2 cells. We further investigated genes regulated by ZFP819 using microarray analysis and chromatin-immunoprecipitation combined with microarray analysis (ChIP-chip) in GC-2 cells. We identified 118 downregulated genes in Zfp819-overexpressing GC-2 cells using microarray analysis. ChIP-chip assay revealed that 1011 promoter sites (corresponding to 262 genes) were specifically enriched in GC-2 cells transfected with Zfp819. Two genes (trinucleotide repeat containing 6b and annexin A11) were commonly found when we compared the data between microarray and ChIP-chip analyses. Consistent with these results, Zfp819 overexpression significantly reduced the transcript levels of the two genes by binding to their promoter regions. Tissue distribution analysis indicated that both genes were predominantly expressed in testis. It has been reported that these two genes function in apoptosis. Collectively, our study provides inclusive information on germ cell-specific gene regulation by ZFP819, which is involved in apoptosis, to maintain the integrity of spermatogenesis.

In mammals, the early embryo travels down the oviduct to the uterus and prepares for implantation. The unique features of preimplantation development include compaction followed by blastocyst formation. This first cell lineage specification involves various proteins including cell polarity regulators, kinases, and transcription factors. In this study, a novel gene named predicted gene 11545 ( Gm11545 ) expressed predominantly in mouse early embryos was identified and characterized at the transcript, protein, cellular, and functional levels. The Gm11545 protein localized to both cytoplasmic and membrane regions of preimplantation embryos. Remarkably, knockdown of Gm11545 led to arrest of mouse embryos at the morula stage and consequent impairment of blastocyst formation. Expression patterns of the key transcription factors critical for early lineage specification, octamer‐binding transcription factor 4 and caudal type homeobox 2, were affected by Gm11545 depletion. Based on the collective findings, we propose that the novel protein identified in this study, Gm11545, is implicated in cell proliferation and cell lineage specification critical for blastocyst formation.—Kim, J., Kim, J., Jeong, J., Hong, S. H., Kim, D., Choi, S., Choi, I., Oh, J. S., Cho, C. Identification of a novel embryo‐prevalent gene, Gm11545 , involved in preimplantation embryogenesis in mice. FASEB J. 33, 11326–11337 (2019). www.fasebj.org

Nuclear RNA homeostasis depends on the balance of transcription, RNA processing, degradation, and transport between the nucleus and cytoplasm. RNA degradation directed by the Integrator, nuclear exosome targeting (NEXT), and nuclear exosome complexes controls the accumulation of aberrant nuclear RNA. Here, we report that Drosophila oogenesis requires the Integrator, NEXT, and nuclear exosome complexes. Depletion of Integrator, NEXT, or nuclear exosome components in Drosophila female germ cells causes infertility and accumulation of 3′ extended snRNAs, promoter upstream transcripts (PROMPTs), and cryptic transcripts. Our data highlight the essential role of nuclear RNA degradation and processing in Drosophila oogenesis and provide a catalog of RNAs whose nuclear levels are regulated by these three complexes. We propose that Integrator, NEXT, and the nuclear exosome support oogenesis by ensuring that inappropriate transcription does not overwhelm the limited supply of proteins that bind, process, and traffic RNA.