Testis-enriched Asb12 is not required for spermatogenesis and fertility in mice.

Background: Members of the ankyrin repeat and SOCS box (Asb) family are expressed abundantly in testes. Some Asb genes/proteins are required for spermatogenesis, but the function of Asb12 during spermatogenesis is not clear. We investigated the physiological role of Asb12 in murine testes.
Methods: The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system was used to generate Asb12-knockout (KO) mice. Histology and immunostaining were done to assess the effects of Asb12 KO on mouse testes and epididymides. Semen quality was analyzed using a computer-assisted sperm analyzer. The terminal deoxynucleotidyl transferase-dUTP nick-end labeling assay was employed to examine testicular apoptosis. Real-time reverse transcription-quantitative polymerase chain reaction (PCR) was conducted to calculate gene transcription levels.
Results: Asb12 was expressed predominantly in murine testes. Immunostaining of Asb12 protein revealed that Asb12 was located specifically in the acrosome of elongated spermatids, which suggested a potential role of Asb12 during spermatogenesis. However, Asb12-KO mice had normal fertility, and no overt difference was detected in testicular morphology, semen quality, or apoptosis when comparing Asb12-KO and Asb12-wild type (WT) mice. Gene expression of several Asb family members was increased significantly in the testes of Asb12-KO mice when compared with that in Asb12-WT mice, which suggested functional compensation from paralogs for Asb12 loss. Conclusions: We demonstrated that Asb12 is not essential for the spermatogenesis and fertility of mice. Our findings will assist researchers in avoiding redundant efforts, and provide a baseline resource for genetic studies on human fertility. 2022 Translational Andrology and Urology. All rights reserved.

Introduction

To ensure the faithful transmission of genetic information to offspring, germ cells undergo dramatic dynamic remodeling throughout spermatogenesis. This process involves the self-renewal of spermatogonial stem cells (SSCs), meiosis, and spermatid maturation (1). Spermatogenesis is an intricate process and coordinated carefully by an elaborate network of genes (2,3). A total of 2,300 genes have been identified as testis-enriched genes in mice (4). Clarifying the role of these genes during spermatogenesis can reveal the molecular mechanisms underlying spermatogenesis, and assist in understanding the etiology of infertility and contraception in males. However, the function of many testis-enriched genes is not known.

Previously, using a gene-knockout (KO) approach mediated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, we generated mice with deleted testis-enriched genes, and our data demonstrated some of them to be crucial for spermatogenesis. For example: T-complex-associated-testis-expressed 1 (TCTE1) is an important axonemal protein that is required for sperm motility (5); F-box protein 47 (FBXO47) is localized specifically in spermatocytes and directs integration between the telomere and inner nuclear envelope during meiotic progression (6); MRN complex interacting protein (MRNIP) is a MRN-interacting protein essential for chromosome synapsis and meiotic homologous recombination (7). On the other hand, we have also shown that several genes [Retinoic acid induced 14 (Rai14), testis expressed 33 (Tex33), Fibronectin type 3 and ankyrin repeat domains 1 (Fank1), and Eukaryotic elongation factor-2 kinase (Eef2k)], previously postulated as crucial factors for spermatogenesis, are dispensable for spermatogenesis and male fertility (8-11).

The ankyrin repeat and suppressor of cytokine signaling (SOCS) box (Asb) protein family contains 18 members in humans and mice. All of them have N-terminal ankyrin (ANK) repeats and a C-terminal SOCS box (12,13). ANK is required for substrate recognition, whereas the SOCS domain interacts with elongin B/C to mediate the ubiquitination and degradation of substrates (14). Recent evidence has revealed a definitive role for the Asb family during spermatogenesis. For example, Asb3, Asb4, Asb8, Asb9, and Asb17 have been reported to be expressed abundantly in mouse testes (15-19); Asb1-KO mice exhibit hypospermatogenesis with an apparent loss of germ cells in seminiferous tubules (15); deficiency of Asb17 in mice can prevent spermatogonial apoptosis through regulation of polyubiquitination of the anti-apoptotic proteins BCL2-like 2 (BCL2L2) and MCL1 apoptosis regulator, BCL2 family member (MCL1) (20). Apart from the roles of the Asb family during spermatogenesis, recent studies have indicated Asb members to be involved in development of the central nervous system (21), myogenesis (22), as well as tumorigenesis (23).

In the present study, we identified Asb12 to be a novel testis-enriched gene in mice. We wished to uncover the role of Asb12 in murine spermatogenesis using an Asb12-KO model. We present the following article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-21-900/rc).

Methods

Ethical approval of the study protocol

This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Bethesda, MD, USA). Animal experiments were approved (license number: 2004020) by the Animal Ethical and Welfare Committee of Nanjing Medical University (Nanjing, China).

Mice

Asb12-KO mice were generated using the CRISPR/Cas9 method according to a protocol reported previously by our research team (8,10). Single guide (sg)RNA was designed to target exon 4 of Asb12: 5'-GTAGATGCTACCACTAGGACAGG-3'. The target sequence was 5'-CCTGTCCTAGTGGTAGCATCTAC-3'. Generation of Cas9 mRNA was done as described previously (24,25). CD-1 mouse zygotes were co-microinjected with sgRNA and Cas9 mRNA. Founders were backcrossed with CD-1 mice to produce heterozygous mice. Pure-background CD-1 Asb12-KO and Asb12-wild-type (WT) mice were used for subsequent experiments. The genotype was identified by polymerase chain reaction (PCR) and subsequent Sanger sequencing using the forward primer 5'-AAGGAGGAGGAGGACACT-3' and reverse primer 5'-AGATGGAAGGTAGATGTTAGC-3'. Experimental animals were maintained under a specific pathogen-free condition at the Animal Center of Nanjing Medical University.

Fertility test

Three paired male (aged 8 weeks) Asb12 (WT and KO) mice were caged, respectively, with WT females (aged 8 weeks) at a ratio of 1:2 for 2 months. The litter size and the number of pups born were recorded.

RNA extraction and real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA from testicular tissues was extracted using TRIzol™ Reagent (Invitrogen, Carlsbad, CA, USA) following manufacturer instructions. Then, RNA was reverse-transcribed into complementary (c)DNA and processed using a real-time PCR system (Applied Biosystems, Foster City, CA, USA) for quantification. 18S rRNA served as an internal control. The primers for each gene are summarized in Table S1.

Histology

Testes or epididymides from Asb12-WT and Asb12-KO mice of age 8–12 weeks were collected and fixed in modified Davidson’s fluid (MDF) for 48 h. Then, tissues were dehydrated in a graded series of ethanol solutions, embedded in paraffin, and sectioned at 4-μm thickness. These sections were rehydrated and stained with hematoxylin and eosin (H&E) or periodic acid Schiff (PAS). Next, we undertook computer-assisted sperm analysis (CASA). Briefly, sperm from Asb12-WT and Asb12-KO mice aged 8–12 weeks were collected from the cauda epididymis and suspended in culture medium. Semen quality was assayed using the Ceros™ II Sperm Analysis System (Hamilton Thorne, Beverly, MA, USA). For ultrastructural analysis, sperm samples from Asb12-WT and Asb12-KO mice aged 8–12 weeks were fixed in 2% (v/v) glutaraldehyde overnight and immersed in 2% (w/v) OsO4 for 2 h. Next, samples were embedded in araldite and sectioned at 80-nm thickness. Images were captured under a transmission electron microscope (JEM-1410; JEOL, Tokyo, Japan).

Immunofluorescence

Sections were deparaffinized and rehydrated, followed by antigen retrieval in sodium citrate buffer as described previously (26,27). After blockade with 1% (w/v) bovine serum albumin for 2 h, samples were reacted with the indicated primary antibodies (Table S2) overnight at 4 °C. Nonimmune immunoglobulin (Ig)G antibodies were used for the negative controls (Figure S1). Subsequently, slides were washed thrice with phosphate-buffered saline (PBS), and then probed with Alexa-Fluor secondary antibodies (Thermo Scientific, Waltham, USA) for 1 h at 37 °C. Finally, sections were counterstained with 4′,6-diamidino-2-phenylindole (Beyotime Institute of Biotechnology, Nantong, China) and subjected to fluorescence analyses under a LSM800 confocal microscope (Zeiss, Oberkochen, Germany).

Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay

Apoptosis was detected using a TUNEL kit (Vazyme, Nanjing, China) following manufacturer instructions. Briefly, paraffin sections were incubated with proteinase K for 15 min before being allowed to react with TUNEL labeling mix buffer for 1 h at 37 °C. Apoptotic cells were counted under a confocal laser scanning microscope (LSM800; Zeiss).

Chromosome spread

Preparation of chromosome spreads was conducted as described previously (28). Briefly, testicular tissues from Asb12-WT and Asb12-KO mice aged 8–12 weeks were digested by collagenase and trypsin for 15 min at 37 °C before immersion in a hypotonic extraction buffer. Then, cell pellets were suspended in sucrose (100 mM) and spread on slides covered with fixative solution. After fixation, samples were washed with PBS and subjected to immunostaining with the indicated primary antibodies (Table S2).

Statistical analyses

Statistical analyses were conducted using Prism 8.0 (GraphPad, La Jolla, CA, USA). Data are the mean ± SD from at least three independent experiments. Biological replicates are indicated in the corresponding figure legends. The unpaired Student’s t-test or chi-square test were used to calculate the differences between Asb12-WT and Asb12-KO mice.

Results

Generation of Asb12-KO mice

ASB12 belongs to the ASB protein family, in which all members have the classic ANK and SOCS domains. Bioinformatic analysis revealed ASB12 to be an evolutionarily conserved protein with conservation of the ANK and SOCS domains among species (Figure S2A). RT-qPCR revealed Asb12 to be expressed abundantly in the mouse testis (Figure S2B). These findings suggested that Asb12 might have a conserved role during spermatogenesis.

To explore the function of Asb12 in vivo, Asb12 mutant mice were generated by CRISPR/Cas9-mediated gene editing which, ultimately, caused a frameshift mutation in exon 4 of Asb12 on the X chromosome by a 1-bp insertion (). This mutation is predicted to result in a truncated protein of 123 amino acids, causing a partial loss of the ANK domain and complete loss of SOCS domains (). The mutation was confirmed by PCR and Sanger sequencing (). Furthermore, using immunostaining of the ASB12 protein in mouse testicular tissue and mature sperm, we observed an obvious location of ASB12 in the acrosome of Asb12-WT (+/Y) mice. In contrast, there was no detectable signal in the acrosome of Asb12 mutant mice (), suggesting an absence of the ASB12 protein in the testes of Asb12 mutant mice. The fluorescence signal in the basal compartment of the seminiferous epithelium was probably non-specific because it was also present in the testes of Asb12 mutant mice (). Thus, we first successfully constructed an Asb12-KO (−/Y) mouse strain. The specific location of ASB12 in mouse sperm suggested that ASB12 may be associated with acrosome maturation during spermatogenesis.

Figure 1

Generation of Asb12-KO mice. (A) CRISPR/Cas9-mediated Asb12 editing (schematic). (B) Analysis of truncated protein in Asb12-KO mice. (C) Sanger sequencing of Asb12-WT and Asb12-KO mice. (D) Immunostaining of Asb12 in the testes of adult Asb12-WT (+/Y) and Asb12-KO (−/Y) mice. Scale bar, 20 µm. (E) Immunostaining of Asb12 in the sperm of Asb12-WT and Asb12-KO mice. PNA served as an acrosome marker. Scale bar, 20 µm. Asb12, Ankyrin repeat and SOCS box protein 12; WT, wild type; Mut, mutation; DAPI, 4′,6-diamidino-2-phenylindole; PNA, Peanut agglutinin.

Normal fertility and semen quality in Asb12-KO mice

Asb12-KO mice developed and survived normally. Asb12-KO males were fertile and led to the production of similar litter sizes to that obtained with Asb12-WT mice (). The testes of Asb12-WT and Asb12-KO mice were comparable in size and weight (). CASA revealed Asb12-KO mice to have a normal concentration, motility, and progressive ratio of sperm compared with those of Asb12-WT mice (). H&E staining of sperm revealed Asb12-KO mice to exhibit normal sperm morphology compared with that of Asb12-WT mice (). Due to the specific location of ASB12 in the acrosome of sperm, we undertook ultrastructural analyses of sperm in Asb12-WT and Asb12-KO mice. Ultrastructurally, Asb12-KO sperm displayed normal morphology with a well-formed acrosome, condensed head, and typical “9 + 2” axoneme (). Moreover, no significant change of progesterone-induced acrosome reaction between WT and KO was observed (Figure S3). We therefore concluded that Asb12 was not required for fertility and semen quality in mice.

Figure 2

Normal fertility in Asb12-KO mice. (A) Fertility test of Asb12-WT and Asb12-KO male mice. n=3 for each group. P>0.05. (B) Gross morphology of the testes of Asb12-WT and Asb12-KO mice. Scale bar, 5 mm. (C) Testis/bodyweight ratio. n=3 for each group. P>0.05. (D-F) CASA of the counts (D), motility (E), and progressive ratio (F) of sperm in Asb12-WT and Asb12-KO mice. M, million. n=3 for each group. P>0.05. (G) H&E staining of sperm in the cauda epididymis of Asb12-WT and Asb12-KO mice. Scale bar, 20 µm. (H) Quantification of (G). n=3 for each group. P>0.05. (I) TEM of the sperm in the cauda epididymis of Asb12-WT and Asb12-KO mice. Arrows indicate axonemes with a “9 + 2” microtubule structure. Scale bar, 0.5 µm. Nu, nucleus; Ac, acrosome.

Normal spermatogenesis in Asb12-KO mice

Testicular morphology was examined by H&E staining. Asb12-WT and Asb12-KO mice exhibited intact seminiferous tubules with all stages of spermatogenic cells (). Moreover, there were no obvious histological changes of the epididymis between Asb12-WT and Asb12-KO mice (). These data suggested that Asb12 deficiency did not have an effect on spermatogenesis.

Figure 3

Histological analysis of testes and epididymides. (A) H&E staining of testis sections in Asb12-WT and Asb12-KO mice. (B) H&E staining of caput epididymides sections in Asb12-WT and Asb12-KO mice. (C) H&E staining of cauda epididymides sections in Asb12-WT and Asb12-KO mice. Scale bar, 50 µm.

Spermatogenesis has three main processes: SSC (spermatogonial stem cell) proliferation, meiosis, and spermiogenesis. To determine precisely the process of spermatogenesis in the testes of Asb12-KO mice, we first undertook PAS staining to analyze the dynamics of acrosome development and nucleus morphology during spermiogenesis. There was no measurable morphological difference in spermatids between Asb12-WT and Asb12-KO mice during spermiogenesis (). In addition, the number of round spermatids and elongating/elongated spermatids was comparable between Asb12-WT and Asb12-KO mice (). Moreover, Lin28, SOX9, and TUNEL signals were used to quantify the number of SSCs, Sertoli cells, and apoptotic cells in testes, respectively: the number of these cell types did not differ between Asb12-WT and Asb12-KO mice (). Finally, the chromosome-spread method was conducted to evaluate meiotic progression. Under co-immunostaining of SCP3 (a marker of the lateral element of the synaptonemal complex) and γH2AX (a marker for DNA double-strand breaks), the subdivided stages of meiotic prophase (leptotene, zygotene, pachytene, and diplotene) could be distinguished readily according to the dynamic distribution of SCP3 and γH2AX (6,7,28). However, marked morphological alteration of SCP3 and γH2AX was not observed in Asb12-KO spermatocytes when compared with that in the WT group (). Taken together, these results indicated that Asb12 was dispensable for mouse spermatogenesis.

Figure 4

Normal spermatogenesis in Asb12-KO mice. (A) PAS staining of spermatogenic stages I–XII in the testes of Asb12-WT and Asb12-KO mice. Scale bar, 20 µm. (B) Quantification of round spermatids (Rs) in (A). n=3 for each group. P>0.05. (C) Quantification of elongating/elongated spermatids (Es) in (A). n=3 for each group. P>0.05. (D) Immunostaining of Lin28 in the testes of Asb12-WT and Asb12-KO mice. Scale bar, 50 µm. (E) Quantification of (D). n=3 for each group. P>0.05. (F) Immunostaining of SOX9 in the testes of Asb12-WT and Asb12-KO mice. Scale bar, 50 µm. (G) Quantification of (D). n=3 for each group. P>0.05. (H) TUNEL assay showing apoptotic cells in the testes of Asb12-WT and Asb12-KO mice. Scale bar, 50 µm. (I) Quantification of (H). n=3 for each group. P>0.05. (J) Co-immunostaining of SCP3 and γH2AX in the spread spermatocytes of Asb12-WT and Asb12-KO mice. Scale bar, 10 µm. (K) Distribution of stage-specific primary spermatocytes in (J). For each group, 500 spermatocytes were included. P>0.05. Rs, Round spermatids; Es, Elongating/elongated spermatids; DAPI, 4′,6-diamidino-2-phenylindole; Lin28, lin‑28 homolog; SOX9, SRY‑box 9; SCP3, synaptonemal complex protein 3; γH2AX, H2A.X variant histone; DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, Terminal deoxynucleotidyl transferase-dUTP nick-end labeling.

Functional compensation from paralogs in the testes of Asb12-KO mice

We undertook real-time RT-qPCR to assess the relative transcription levels of the 18 Asb genes in the testes of Asb12-WT and Asb12-KO mice. Interestingly, expression of Asb-1,2, 3, 4, 5, 7, 8, 9, 11, 14, 15, 17, and 18 was increased significantly in the testes of Asb12-KO mice compared with that in the testes of Asb12-WT mice (). The relative transcription level of Asb12 was reduced markedly in the testes of Asb12-KO mice (), which suggested that Asb12 mutation could cause a decay of the encoded mRNA. This finding strongly suggests that other members of the Asb family may compensate for Asb12 deficiency.

Figure 5

Functional compensation from paralogs in the testes of Asb12-KO mice. Real-time reverse transcription-quantitative polymerase chain reaction of the transcription levels of the 18 Asb genes in the testes of Asb12-WT and Asb12-KO mice. Gene expression was normalized to that of 18s rRNA. n=3 for each group. *, P<0.05. Asb, ankyrin repeat and SOCS box.

Discussion

A gene microarray study by Schultz et al. uncovered >2,300 testis-enriched genes in the mouse genome. Using the gene-KO approach in mice, we and other research teams have revealed many testis-enriched genes that are required for spermatogenesis and male fertility (5-7,29-31). Nevertheless, several testis-enriched genes that had been postulated previously to be essential factors for spermatogenesis have been shown to be unnecessary for spermatogenesis in KO animal models (32). Although gene-KO mice with no obvious reproductive phenotypes have rarely been reported, the genetic and phenotypic information must be disseminated to the scientific community to prevent redundant efforts by other research teams. In 2017, Castaneda et al. shared information on 54 testis-enriched genes that are not essential for spermatogenesis or fertility in mice via a genome-engineering approach. Thereafter, several large-scale screenings of non-essential testis-enriched genes were published (33,34).

Our research team has been using the CRISPR/Cas9 method to produce mice that delete testis-enriched genes. Here, we investigated the effects of the loss of a testis-enriched gene, Asb12, on spermatogenesis and fertility in mice using CRISPR/Cas9-mediated gene editing. We discovered that, despite the evolutionary conservation of Asb12 in vertebrates, it is not essential for spermatogenesis and male fertility. Although such functional redundancy renders Asb12 irrelevant as a target for contraception, the restricted expression of ASB12 in the sperm acrosome may enable it to serve as a potential biomarker for non-obstructive azoospermia.

A reasonable explanation for Asb12-KO mice displaying normal spermatogenesis and fertility may be functional redundancy. Genetic compensation upon gene KO is a common phenomenon among species. Paralogous genes with overlapping functions may compensate for the loss of one gene (35,36). For instance, KO of Ubqln3 in mouse testes induces significant upregulation of expression of Ubqln1, Ubqln2, and Ubqln4, whereas KO of UbqlnL provokes a marked increase in expression of Ubqln1 and Ubqln4 in testes (37,38). Likewise, in our study, a loss of Asb12 in mouse testes elicited a dramatic increase in expression of several Asb members. To overcome functional redundancy, generation of double-KO (or even triple-KO) models is needed to eliminate the effects of functional compensation from paralogs.

Conclusions

In this study, we generated a murine Asb12-KO model. Although Asb12 was shown to be a testis-enriched gene in mice, Asb12 itself had no obvious effects on spermatogenesis and male fertility in mice. The phenotypic information of Asb12-KO mice we have provided will assist other researchers in prioritizing their resources to focus on genes that are crucial for male fertility.

The article’s supplementary files as