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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 1  |  Page : 18-23

LncRNA4667 is dispensable for spermatogenesis and fertility in mice


1 Department of Human Anatomy, Histology and Embryology, School of Basic Medicine, Qingdao University, Qingdao 266071, Shandong, China
2 Institute of Embryo-Fetal Original Adult Disease, International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, China
3 Shanghai Key Laboratory for Reproductive Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, China
4 Department of Human Anatomy, Histology and Embryology, School of Basic Medicine, Qingdao University, Qingdao 266071, Shandong; Institute of Embryo-Fetal Original Adult Disease, International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030; Institute of Reproductive Medicine, School of Medicine, Nantong University, Nantong 226001, China

Date of Submission18-Feb-2019
Date of Web Publication11-Apr-2019

Correspondence Address:
Fei Sun
Institute of Reproductive Medicine, School of Medicine, Nantong University, 19 Qixiu Road, Nantong 226001
China
Ning Song
Shanghai Key Laboratory for Reproductive Medicine, School of Medicine, Shanghai Jiao Tong University, 228 South Chongqing Road, Shanghai 200030
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.255985

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  Abstract 


Objective: Spermatogenesis is a complex process which is of vital importance for sexual reproduction. In many studies of spermatogenesis, the mRNAs, protein-coding genes, as well as small noncoding RNAs (ncRNAs) have been well characterized. However, there remain numerous questions despite previously characterized molecular mechanisms. Long ncRNAs (lncRNAs) are a relatively new addition to our knowledge of ncRNAs. Limited studies have examined the function of lncRNAs in spermatogenesis. Therefore, we selected a testis-specific lncRNA, lncRNA4667, to analyze its potential role in spermatogenesis and male fertility.
Methods: In situ hybridization and quantitative reverse transcription polymerase chain reaction analyses were used to confirm testis-specific expression of lncRNA4667. LncRNA4667 knockout mice were generated using CRISPR/Cas9 technology. Histology, sperm counts, sperm motility, body parameters, and fertility were compared between wild-type and knockout mice (n = 8/group).
Results: Expression analysis showed that lncRNA4667 was testis specific and localized to round spermatids in seminiferous tubules of adult mouse testes. Mice homozygous for a null mutation of lncRNA4667 displayed normal spermatogenesis and fertility compared with wild-type mice.
Conclusions: These data indicate that lncRNA4667 is dispensable for spermatogenesis and fertility in mice, and the localization of lncRNA4667 makes it a useful marker for the identification of round spermatids in mice.

Keywords: Long Noncoding RNA; Male Infertility; Spermatogenesis


How to cite this article:
Dai YB, Lin Y, Song N, Sun F. LncRNA4667 is dispensable for spermatogenesis and fertility in mice. Reprod Dev Med 2019;3:18-23

How to cite this URL:
Dai YB, Lin Y, Song N, Sun F. LncRNA4667 is dispensable for spermatogenesis and fertility in mice. Reprod Dev Med [serial online] 2019 [cited 2019 Nov 22];3:18-23. Available from: http://www.repdevmed.org/text.asp?2019/3/1/18/255985




  Introduction Top


Spermatogenesis is a complex multistage process, in which spermatogonial stem cells (SSCs) ultimately differentiate into haploid spermatids, and includes SSC renewal, meiosis of spermatocytes, and the differentiation of haploid spermatids.[1],[2] Noncoding RNAs (ncRNAs) have been grouped into two categories based on the length of their mature products: small ncRNAs and long ncRNAs (lncRNAs).[3] Transcription of ncRNAs in the mammalian genome occurred at higher levels than mRNA transcription.[4] Many studies have revealed protein-coding mRNAs, as well as small ncRNAs (e.g., microRNAs and piwi-interacting RNAs), that are essential for regulating mammalian reproduction.[5],[6] Recent developments in genome sequencing technology have rapidly expanded the ncRNAs family.

LncRNAs, longer than 200 nucleotides, are a relatively new addition to the ncRNA family.[7] The lncRNAs play prominent roles in the regulation of many biological processes, such as gene silencing, angiogenesis, cell proliferation, gonadogenesis, and sexual determination.[8],[9],[10],[11],[12] Recent studies found that numerous lncRNAs are highly expressed in the testis,[13],[14] suggesting that they may play a crucial role in spermatogenesis. The lncRNA-testicular cell adhesion molecule 1 (lncRNA-Tcam1), specifically transcribed from the enhancer region of Tcam1, was shown to be important for the immune response during spermatogenesis.[15],[16] In addition, deficiencies of some key lncRNAs may contribute to the loss of germ cells and male infertility.[17],[18],[19] However, few lncRNAs have been well characterized in terms of function and molecular mechanisms. In particular, lncRNAs with testis-specific expression should be given more attention with regard to potential biological functions in spermatogenesis.

From our previous study of lncRNA expression patterns in the mouse testis by RNA-Seq, we selected an lncRNA that was enriched in the testis, known as lncRNA4667. The corresponding DNA locates in chr13:23396074–23397418. This study aimed to determine the in vivo expression and function of lncRNA4667during mouse spermatogenesis. Using in situ hybridization (ISH) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses, we showed that lncRNA4667 is a mouse testis-specific lncRNA (Tslrn) selectively expressed in round spermatids, making it a useful marker for the identification of round spermatids in seminiferous tubules. We prepared lncRNA4667 knockout mice using the CRISPER/Cas9 approach. However, mice homozygous for a null mutation of lncRNA4667 displayed normal spermatogenesis and fertility. Our finding suggests that lncRNA4667 is not essential for spermatogenesis and the fertility of male mice.


  Methods Top


Experimental mice

The experimental model was established using C57BL/6 mice, which were provided food and water ad libitum and were kept in a 14 h light/10 h dark cycle. All animal experiments in this study were approved by the Ethics Committee of the International Peace Maternity and Child Health Hospital (GKLW 2016-31).

RNA extraction, quantitative reverse transcription polymerase chain reaction, and 5' and 3' rapid amplification of cDNA ends

Testicular RNA was extracted using TRIzol reagent (Ambion, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. The RT-qPCR for lncRNA4667 expression was performed on a 7500 system (Applied Biosystems, Foster City, CA, USA) using an SYBR Premix110 EX Taq II kit (TaKaRa Bio, Dalian, China), following the manufacturer's protocols. Glyceraldehyde 3-phosphate dehydrogenase was used as the reference gene. Primers sequences used were as follows: Forward: 5′-GACTTCACGGACCTCGACAG-3′; Reverse: 5′-GATCCCGCCTCTCTACTGGA-3′. Full-length cDNA sequences were obtained by rapid amplification of cDNA end (RACE) using the SMARTer RACE 5′/3′ Kit (TaKaRa Bio, Dalian, China) according to the manufacturer's instructions. The RACE primer sequences were as follows: 5′-Race: 5′-AGACATAAGAGCTGCTTCTACGAC-3′; 3′-Race: 5′-AACTTTGTCATCAAATATCTGTAGA-3′.

Production of lncRNA4667 knockout mice

We used CRISPR/Cas9 technology to generate lncRNA4667 knockout mice. Guide RNAs (gRNAs) were designed on both sides of lncRNA4667. Both target gene gRNAs, gRNA1 and gRNA2, were injected along with Cas9 protein into the pronucleus of C57BL/6 one-cell stage embryos, which were transferred to pseudopregnant females to develop. Thirty transgenic offspring (founders) were identified by the presence and/or absence of the target gene by PCR using the following primers: P1: 5′-CGCAGTCGGAGACCTTGT-3′; P2: 5′-AACGGAGAAGCTCACTAAATGGA-3′. We used RT-qPCR to investigate lncRNA4667 expression in testes from wild-type and knockout mice.

In situ hybridization

To investigate the cell-specific distribution of lncRNA4667 in wild-type and lncRNA4667 knockout mouse tissues, ISH was performed using the RNAscope 2.5 LS Reagent Kit (Advanced Cell Diagnostics [ACD], Hayward, CA, USA) and mouse lncRNA4667 probe (ACD catalogue number 457111-C2, NCBI reference sequence gi / 372099097:23396075-23397418) according to the manufacturer's protocol. The signal was visualized with Fast Red chromogen and the slides were counterstained with hematoxylin. Signals were scanned using a microscope-mounted digital camera (Olympus model DP74, Tokyo, Japan).

Sperm counts and motility

Sperm samples were assessed by unilateral removal of the epididymis from 70-day postpartum (dpp) knockout and wild-type mice. Epididymal tissue was cut several times and then placed in 1 mL of medium containing 0.5% bovine serum albumin (Yeasen, Shanghai, China) at 37°C with 5% CO2 for 30 min. Then, we used a computer-assisted sperm analyzer (CASA, Cyto-S, VideoTesT, Russia) to examine sperm counts and motility.

Histological examination

For histology, 70-dpp knockout and wild-type mice were euthanized by cervical dislocation. Epididymides and testes were immediately fixed in Bouin's solution, embedded in paraffin wax, and sectioned at 5-μm thickness for hematoxylin and eosin staining.

Statistical analysis

The data were analyzed using GraphPad Prism 5.01 (GraphPad Prism, La Jolla, CA, USA). Student's t-tests and one-way analysis of variance were applied to examine the differences between paired means of variables. The results are shown as the mean ± standard error of the mean. P < 0.05 was considered statistically significant.


  Results Top


Analysis of lncRNA4667 expression in adult mice

From our previous RNA-Seq analysis, we selected one lncRNA, lncRNA4667, which was expressed exclusively in the testis of adult mice and located on mouse chromosome 13. Using RT-qPCR, we confirmed that lncRNA4667 was specifically expressed in the mouse testis [Figure 1]a. We examined the expression of lncRNA4667 in a panel of mouse tissues (testis, brain, heart, lung, liver, kidney, and epididymis) and found that lncRNA4667 was only expressed in the testis [Figure 1]a.
Figure 1: Analysis of lncRNA4667 expression in adult mice (70-day postpartum). (a) LncRNA4667 expression levels in adult mouse organs (testis, brain, heart, lung, and epididymis). (b) PCR products of lncRNA4667 from the 5'- and 3'-RACE procedures. (c) Expression of lncRNA4667 in mouse testis by in situ hybridization. Hybridization signals (red) for lncRNA4667 and negative control are shown. Scale bar = 50 μm. *P < 0.001. RS: Round spermatids; lncRNA: Long noncoding RNA; PCR: Polymerase chain reaction; RACE: Rapid amplification of cDNA end.

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We used 5′- and 3′-RACE to detect the actual length of lncRNA4667 [Figure 1]b. The total length of lncRNA4667 was 1344 bp, which was consistent with the results from our RNA-seq analysis. The sequence information is listed in supplemental materials.

We used ISH to identify the testicular cell types expressing lncRNA4667 in adult mice (70-dpp) [Figure 1]c. LncRNA4667 was detected in all seminiferous tubule sections and was highly expressed in round spermatids, but not in Sertoli and interstitial cells.

Preparation and verification of the lncRNA4667 knockout model

We generated lncRNA4667 knockout mice by the CRISPR/Cas9 system [Figure 2]a to study the function of lncRNA4667 during spermatogenesis. From thirty founders, we obtained 8 (26.7%) mice with complete deletion of lncRNA4667. We then confirmed the absence of lncRNA4667 [Figure 2]b,[Figure 2]c,[Figure 2]d. RT-qPCR analysis showed a marked reduction in RNA levels in the knockout mouse testes [Figure 2]c. Examination by ISH revealed that the expression of lncRNA4667 was totally abolished in knockout testes [Figure 2]d. Overall, these analyses suggested the successful generation of the lncRNA4667 knockout model.
Figure 2: Creation and verification of lncRNA4667 knockout mice. (a) Schematic of the knockout strategy of lncRNA46676 using the CRISPR/Cas9 system. (b) PCR was used to identify genotypes. Knockout: #1 and #2, heterozygote: #3 and #4, wild-type: #5. (c) Quantitative RT-PCR of lncRNA4667 expression from testes of wild-type, heterozygote, and knockout males. (d) Localization of lncRNA4667 by in situ hybridization in testis sections from negative control and knockout mice (70 days postpartum). Scale bar = 50 μm. *P < 0.001. lncRNA: Long noncoding RNA; RT: Reverse transcription; PCR: Polymerase chain reaction.

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LncRNA4667 knockout males are fertile

We investigated the effect of lncRNA4667 absence on spermatogenesis in the knockout model. There were no obvious differences in the gross examination of testes and epididymides between lncRNA4667 knockout and wild-type control males [Figure 3]a. Likewise, CASA analysis showed no difference in total sperm motility and progressive motility between knockout and wild-type mice [Figure 3]b and [Figure 3]d. Testicular and epididymal histology suggested no differences between these groups [Figure 3]c. The number of pups per litter produced by mating knockout male mice with wild-type females was similar to those produced by wild-type mating pairs [Figure 3]e. In addition, body length and weight, testis size, and sperm counts were equivalent in knockout and wild-type males [Table 1]. Overall, these results suggested that loss of lncRNA4667 in mice does not influence fertility or spermatogenesis.
Figure 3: Normal spermatogenesis in lncRNA4667 knockout mouse testes (70-day postpartum). (a) Gross comparison of testes and epididymides from adult knockout and wild-type mice. (b) Total sperm motility found in knockout and wild-type mice. (c) Histological comparison of seminiferous tubules and epididymides (caput, cauda, and corpus) from knockout and wild-type mice. (d) Analysis of sperm progressive motility found in knockout and wild-type mice. (e) The number of pups per litter from knockout (n = 8) and wild-type (n = 8) male breeders. Scale bar = 50 μm. lncRNA: Long noncoding RNA.

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Table 1: Reproductive data and body parameters of lncRNA4667 knockout male mice

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  Discussion Top


While miRNAs, piRNAs, and small interfering RNAs have been well studied during germline development,[6],[20],[21] the possible functions of the more recently identified lncRNAs are poorly understood. Potential functions of lncRNAs have been predicted based on their tissue-specific localization, with many lncRNAs most abundant in the testis.[22] The lncRNAs can interact with many biological structures, such as mRNA, DNA, microRNA, RNA-binding proteins, and chromatin.[23],[24],[25] The investigation of Tslrn is a crucial first step to understand their biological function and potential mechanisms in spermatogenesis. This study examined the role of lncRNA4667, which was found to be testis enriched in our previous RNA-seq analysis. We have successfully generated a lncRNA4667 knockout mouse model by CRISPR/Cas9 to investigate its role in spermatogenesis and male fertility.

For the first time, our study describes the expression and subcellular localization of lncRNA4667. We found that lncRNA4667 was testis specific and expressed in all seminiferous tubules of adult mouse testes. In particular, lncRNA4667 was specifically enriched in round spermatids, whereas no expression was detected in Sertoli and interstitial cells. The expression pattern of lncRNA4667 suggests that it may provide a useful molecular marker for the identification of round spermatids in mice. Previous studies found that, in lncRNA knockout models in Drosophila, nearly one-third of the groups exhibited a loss of male fertility, suggesting that lncRNA may play an important role in male reproduction.[19],[26] In the current study, the subcellular localization of lncRNA4667 suggested that it may contribute to the regulation of differentiation during the later stages of murine spermatogenesis.

We created the lncRNA4667 knockout mouse model to investigate the potential function of lncRNA4667 in male reproduction. The absence of lncRNA4667 transcripts in round spermatids of knockout mice confirmed the gene disruption in this model. We found that male mice lacking lncRNA4667 have normal spermatogenesis. Furthermore, all lncRNA4667 knockout male mice exhibited normal sperm counts and motility, as well as normal fertility. These results indicated that lncRNA4667 is not essential for murine spermatogenesis. The haploid round spermatids, where lncRNA4667 was localized, constitute nearly 45% of all cells in the testis.[27] The lncRNAs tend to be more highly expressed in round spermatids compared to other germ cells or somatic cells.[28] We hypothesize that some lncRNAs may compensate for the loss of lncRNA4667 in round spermatids in the mouse testis. Another abundant and highly conserved lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (Malat1), was expressed in sperm and interstitial cells of the testis. Previous studies showed that Malat1 knockout mice also exhibited normal fertility.[29] Other lncRNAs are expressed in the male germline, such as Tslrn1, which was highly expressed in pachytene spermatocytes. However, mice carrying a Tslrn1 deletion produced normal litter sizes compared with wild-type mice.[30] These findings indicate that not all Tslrn are essential for spermatogenesis. However, additional functional exploration of these lncRNAs in spermatogenesis is required.

In conclusion, our study showed for the first time that a Tslrn, lncRNA4667, was specifically expressed in round spermatids and may provide a useful marker to identify germ cells. The deletion of lncRNA4667 did not influence the process of spermatogenesis. We propose that other unidentified lncRNAs in round spermatids may compensate for the disruption of lncRNA4667 function, which needs further investigation.

Financial support and sponsorship

This work was supported by the National Key Research and Development Program of China (Grant No. 2018YFC1003500 to FS) and the National Natural Science Foundation of China Grants (Grant No. 81430027 and 81671510 to FS, 81501307 to Ning Song).

Conflicts of interest

There are no conflicts of interest.



 
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