SPATS1 (spermatogenesis-associated, serine-rich 1) is not essential for spermatogenesis and fertility in mouse

Carlos A Capoano; Luis Adrián Ortiz-Laquintana; Rosana Rodríguez-Casuriaga; Geraldine Schlapp; María Noel Meikle; Ana Paula Mulet; Martina Crispo; Ricardo Benavente; Adriana Geisinger

doi:10.1371/journal.pone.0251028

. 2021 May 4;16(5):e0251028. doi: 10.1371/journal.pone.0251028

SPATS1 (spermatogenesis-associated, serine-rich 1) is not essential for spermatogenesis and fertility in mouse

Carlos A Capoano ¹, Luis Adrián Ortiz-Laquintana ¹, Rosana Rodríguez-Casuriaga ¹, Geraldine Schlapp ², María Noel Meikle ², Ana Paula Mulet ², Martina Crispo ², Ricardo Benavente ³, Adriana Geisinger ^1,^4,^*

Editor: Suresh Yenugu⁵

PMCID: PMC8096103 PMID: 33945571

Abstract

SPATS1 (spermatogenesis-associated, serine-rich 1) is an evolutionarily conserved, testis-specific protein that is differentially expressed during rat male meiotic prophase. Some reports have suggested a link between SPATS1 underexpression/mutation and human pathologies such as male infertility and testicular cancer. Given the absence of functional studies, we generated a Spats1 loss-of-function mouse model using CRISPR/Cas9 technology. The phenotypic analysis showed no overt phenotype in Spats1^-/- mice, with both males and females being fertile. Flow cytometry and histological analyses did not show differences in the testicular content and histology between WT and knockout mice. Moreover, no significant differences in sperm concentration, motility, and morphology, were observed between WT and KO mice. These results were obtained both for young adults and for aged animals. Besides, although an involvement of SPATS1 in the Wnt signaling pathway has been suggested, we did not detect changes in the expression levels of typical Wnt pathway-target genes in mutant individuals. Thus, albeit Spats1 alteration might be a risk factor for male testicular health, we hereby show that this gene is not individually essential for male fertility and spermatogenesis in mouse.

Introduction

Mammalian spermatogenesis involves the execution of three successive gene expression programs: the mitotic proliferation of spermatogonia (i.e. meiotic precursor cells), meiotic divisions, and spermiogenesis (i.e. the differentiation process of round spermatids, which are the outcome of meiosis, into sperm) [1]. Due to the continuity and asynchronicity of the spermatogenic process [2], the three gene expression programs coexist within adult testes. Several studies have shown that the transcriptomes of meiotic and post-meiotic cells are extremely complex, and exhibit the expression of a very high number of specific genes (e.g. [3–5]).

The alteration of spermatogenic gene expression programs is at the basis of numerous pathologies, including infertility [6–11] and testicular cancer [12]. However, owing to some intrinsic obstacles–namely the high cell heterogeneity of testicular tissue, and the lack of reliable in vitro cultures [13–16]–the studies on the molecular groundwork of spermatogenesis, including the identification of those genes that are essential for fertility, have been hampered. To assess the relevance of each of the reproductive genes/proteins, their functional characterization is required.

Spats1 (‘‘spermatogenesis-associated, serine-rich 1”; firstly called Srsp1) was previously identified in our laboratory as a testis-specific gene in rat [17]. The protein was first detected in embryo testes at 17.5–18 days post-coitum (dpc), where it was evident in gonocytes and peritubular myoid cells. Then it is accumulated during postnatal testis development reaching its maximum expression levels at 21 days postpartum (dpp) coincidentally with the pachytene stage, while signal intensity decreased in adult testes. Overall, the highest SPATS1 signal was observed in spermatocytes I (i.e. meiotic prophase), although relatively high protein levels were detected in Sertoli cells, spermatogonia, and myoid cells as well [18]. RNAseq studies showed that for mouse, the highest Spats1 expression levels among different spermatogenic cell types were in primary spermatocytes as well [19]. We have also shown that SPATS1 is a highly phosphorylatable protein [18]. Moreover, SPATS1 is highly conserved along the evolution of metazoans, which allows to suspect that this protein could have an important, conserved role in testis [18].

More recently, a couple of reports have suggested a link between SPATS1 and male fertility. In one of these reports, the expression levels of Spats1 were notably decreased in men with severely impaired spermatogenesis [20]. In the other—a genome-wide association study (GWAS)—Spats1 was revealed as a potential candidate gene related to sperm quality in Holstein-Fresian bulls [21]. Furthermore, an exome-wide sequencing study has proposed a possible association between Spats1 mutation and the development of human seminomas, which are the most common type of testicular cancers [22]. On the other hand, an existing report has suggested a link between SPATS1 and the canonical Wnt signaling pathway [23], a key regulatory pathway with roles in the control of diverse developmental processes and in cancer (e.g. [24–27]).

Nevertheless, no functional studies towards the elucidation of the role of Spats1 in testis have been performed so far. As an attempt to contribute to the characterization of its function, we generated Spats1 loss-of-function mice by means of CRISPR/Cas9 technology. Our results show that Spats1 is not individually essential for mouse male fertility.

Materials and methods

Evolutionary conservation

For homology searches of mouse Spats1, we used the Mus musculus Spats1 mRNA sequence from Genbank (NCBI Reference Sequence: NM_027649.3; this sequence corresponds to transcript ENSMUST00000024731.8 from Ensembl project database). For protein comparisons, the employed protein sequence was AAI31910 (A2RRY8 in the Uniprot database), of 269 amino acids. Sequence homology searches were made with the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi), using blastp and tblastn tools. The comparisons were conducted alternatively against the entire database, against vertebrates exclusively, or excluding mammals (according to the parameters selected in each case). Sequence alignments were performed with ClustalW. A highly resolved tree for phylogenetic relationship between SPATS1 along the evolution of metazoans was built in MEGA (Molecular Evolutionary Genetics Analysis) [28].

For Spats1 tissue-specific expression pattern analysis in different species, we collected all the available information in Expression Atlas (https://www.ebi.ac.uk/gxa/home) and NCBI databases.

Animals

All animal procedures to generate the KO mice were performed at the SPF animal facility of the Transgenic and Experimental Animal Unit, Institut Pasteur de Montevideo (UATE, IPMontevideo). Experimental protocols were accordingly approved by the Ethics Committee for Animal Use (“Comisión de Ética en el Uso de Animales” [CEUA], protocol number 001–13), in accordance with national law 18,611 (Uruguay) and international animal care guidelines (Guide for the Care and Use of Laboratory Animals) (NRC, 1996). B6D2 F1 hybrid mice to generate the KO line were bred at the UATE.

All subsequent experimental animal procedures were performed at Instituto de Investigaciones Biológicas Clemente Estable (IIBCE, Montevideo, Uruguay), also in accordance with national law 18,611, and following the recommendations of the Uruguayan National Commission of Animal Experimentation (“Comisión Nacional de Experimentación Animal” [CNEA], approved experimental protocol 004/09/2011).

Male mice were humanely euthanized by cervical dislocation. Testes were measured and weighed individually, after removal of the tunica albuginea.

Production of CRISPR/Cas9 knockout mice

Potential cleavage sites were retrieved with Custom gRNA software (Thermo Fisher Scientific, Waltham, MA, US), allowing for up to 3 mismatches in the single guide RNA (sgRNA) target sequence. The following sgRNA target, within the 3rd exon of Spats1, was chosen because it did not show any off-target sequences in the genome of M. musculus: TTCGCTGCCTGAAATCCCAAAGG (the protospacer adjacent motif [PAM] sequence is in bold characters). The sgRNA and Cas9 mRNA were ordered from Invitrogen (Carlsbad, CA, US). Validation of sgRNA/CRISPR cleavage activity was done by means of GenArt Genomic Cleavage Detection Kit (Invitrogen) following the instructions of the manufacturer, and using transfected NIH 3T3 cells.

Following validation of the sgRNAs, a mix of 50 ng/μL Cas9 mRNA and 20 ng/μL sgRNA diluted in microinjection buffer were microinjected into the cytoplasm of one-cell B6D2 F2 embryos. Approximately 200 zygotes per assay were microinjected, using a micromanipulator system (Transfermann NK2, Eppendorf, Germany). The microinjected zygotes were immediately transferred into the oviduct of recipient pseudopregnant females of the same strain (20–25 embryos/female). Full-term pups were naturally delivered 19–21 days later. Three backcrosses to C57BL/6J were done to obtain KO and WT littermates for the different experiments.

Genotyping

At 21 days postpartum, immediately after weaning, the pups were appropriately numbered and approximately 0.5 cm of each tail tip was biopsied and used for genotyping. Genomic DNA was extracted from each tail tip with the GeneJET Genomic DNA Purification Kit (Thermo Fisher), and the region flanking the target site (465 bp) was amplified by standard PCR. The designed primers (Spats1-E3-Forward and Spats1-E3-Reverse) are listed in Supplementary S1 Table. PCR bands were extracted from agarose gel by means of the GeneJET Gel Extraction and DNA Cleanup Micro Kit (Thermo Fisher), and direct sequencing of the PCR products by Sanger sequencing from both ends was performed.

Fertility test

Spats1^-/- males (45 dpp) were caged with Spats1^-/- females, in duplicate experiments for up to three months. In another experiment, Spats1^-/- males (45 dpp) were caged each with 3 sexually mature WT females for the same time; six replicate experiments were performed. In parallel, Spats1^-/- females were mated with 2 sexually mature WT males each for the same time lapse; four replicates were done in this case. The number of litters and pups from each pregnant female was recorded at birth. Experimental animals were compared with littermate controls when possible, or otherwise with age-matched non-littermate controls from the same colony.

For fertility tests of one-year old mice, Spats1^-/- or WT males were mated with 3 WT females for three months in duplicate experiments.

Western blot

Decapsulated testes were minced with a Teflon homogenizer and resuspended in Laemmli buffer. We loaded 15 μg of protein lysate per lane. Extracts were separated by SDS/PAGE (12% polyacrylamide), followed by liquid transfer to Protran nitrocellulose (Schleicher & Schuell, Germany). The membranes were blocked o.n. in TBST (Tris-buffered saline with 0.1% Tween-20) containing 10% nonfat milk, probed for 2 h with anti-SPATS1 primary antibody (sc-139435, 1:1,000; Santa Cruz Biotechnology, Dallas, TX, US) diluted in TBST, followed by 1 h incubation with anti-rabbit IgG HRP-linked secondary antibody (Pierce-Thermo Fisher, 1:30,000). Signal was detected by means of Supersignal West Pico Chemiluminescent Substrate Kit (Pierce-Thermo Fisher). Probing with anti-β-actin (ab8227, Abcam, Cambridge, UK) was used as a control for equal loading.

Histological analysis

Whole testes were immersed in a fixative solution containing 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 6.9), and left overnight. Samples were cut into smaller pieces, washed with 0.1 M phosphate buffer (6 x 10 min), postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h, and washed again with the same buffer solution (3 x 10 min). Samples were dehydrated in ethanol and embedded in Epon (Durcupan, Fluka/Sigma-Aldrich, St Louis, MO, US) according to conventional procedures. Then, 250 nm sections were cut using a Power Tome XL ultra-microtome (Boeckeler Instruments, Tucson, AZ, US), stained with toluidine blue, examined by bright field microscopy under an Olympus FV300 microscope (Olympus, Tokyo, Japan), and photographed with an Olympus DP70 digital camera by means of DPController v. 1.1.1.65 software.

Analysis of testicular cell populations by flow cytometry (FCM)

Testes from 45-day-old mice were dissected into 96 mm glass Petri dishes containing ice-cold separation medium (10% v/v FCS in Dulbecco’s Modified Eagle’s medium, DMEM, with high glucose and L-glutamine), and cut into 2–3 mm³ pieces after removal of the tunica albuginea. Three to four of these pieces were immediately placed into a disposable disaggregator Medicon with 50 μm separator mesh (BD Biosciences, San Jose, CA, US) and processed in a Medimachine device (BD) as previously instructed [29, 30]. Afterwards, the cells were fixed in 70% ethanol o.n. at 4°C, centrifuged for 5 min at 1,000 rpm, and the pellet was resuspended in 500 μL PBS. After 5 min at room temperature, propidium iodide solution (PI, Sigma-Aldrich; 1 mg/mL) and RNase were added to the cell suspension at a final concentration of 50 μg/mL each, at 0°C in the dark, and staining was allowed to proceed for 10 min at 0°C in the dark before analysis.

Cells were analyzed with a FACSVantage flow cytometer (BD) equipped with a water-cooled argon ion laser (Coherent, Innova 304) tuned to emit at 488 nm of excitation wavelength. Laser power was set to 100 mW and the fluorescence emitted from PI was collected in FL2 using a 575/26 band pass filter. A 70 μm nozzle was selected to perform FCM measurements. DNA QC particles (BD) were used to optimize fluorescence detection as well as to check instrument linearity and doublet discrimination performance. Analysis of the following parameters was carried out with CELLQuest software (BD): forward scatter (FSC-H), side scatter (SSC-H), pulse-area or total emitted fluorescence (FL2-A), and pulse-width or duration of fluorescence emission (FL2-W). Dot plots of FL2-A vs time were used as a control of fluorescence emission during sample analysis. Doublets were excluded from the analysis using dot plots of FL2-A vs FL2-W. The quantifications of the cell populations were also performed with CELLQuest. Half a testicle was used for each analysis, and 4 animals of each type were analysed in parallel.

Sperm motility and morphology

For motility analysis, the cauda epididymis of either 50 dpp or 1 year old mice were cut, and sperm were allowed to swim out for 15 min in 500 μL of HTF modified medium (50% solution A [200mM NaCl, 0.2% glucose, 5 mM KCl, 0.7 mM Na₂HPO₄.H₂O, 1 mM C₃H₃NaO₃, 0.12 mg/mL penicillin G, 0.1 mg/mL streptomycin, 3.5 mM CaCl₂.2H₂O, 1 mM MgCl₂, 60% sodium lactate]; 50% solution B [50 mM NaHCO3]; 0.3% BSA). For each animal, one epididymis was incubated at 37°C for 10 min in a CO₂ incubator before analysis, while the other was incubated for 60 minutes to promote in vitro capacitation. Five μL per slide were loaded, and observed under a Nikon E200 Eclipse microscope (Nikon Instruments, Tokyo, Japan). Sperm motility was assessed by computer-assisted sperm analysis (CASA). Movement characteristics were evaluated using an automated analyzer (SCA-Microoptics, Barcelona, Spain), with parameter settings for mouse. Semen from 3 KO and 3 WT individuals was evaluated in each case.

Sperm morphology was evaluated with an Olympus FV300 microscope under differential interference contrast (DIC) microscopy, using FluoView software (Olympus). For 50 dpp males we used 6 WT and 6 KO individuals, while for 1 year old males, 5 individuals of each type were employed. At least 200 sperm per individual were analyzed. Sperm heads were classified as morphologically normal or anomalous. In turn, anomalous heads were categorized as: without hook, banana-shaped, and “others”, based on the categorizations of other authors [31, 32].

qRT-PCR

Total RNA was extracted from WT and KO testes using PureLink RNA Mini Kit (Ambion-Thermo Fisher, Foster City, CA, US). RT and qPCR experiments were performed with the Power SYBR Green Cells-to-Ct Kit (Ambion-Thermo Fisher), in a qPCR CFX96 Touch™ Real-Time PCR Detection System (BioRad, Hercules, CA, US). For retrotranscription, 20 ng RNA were used in 50-μL final volume for 1 h at 42°C in accordance with the instructions from the manufacturer, and 2 μL cDNA were used for each PCR reaction. All the reactions were performed with three biological replicas. A calibration curve was built with serial dilutions of Ppp1cc gene (protein phosphatase 1, catalytic subunit, gamma isozyme), which had been previously validated as a good housekeeping gene for mouse testis [19]. Absolute expression values of each gene in arbirtrary units were obtained according to the calibration curve defined with the Ppp1cc gene. Additionally, crossed validation was performed with Tax1bp1 (human T-cell leukemia virus type I-binding protein 1), a second gene whose usefulness as a calibrator gene had been previously demonstrated as well [19]. The primer sequences are listed in S1 Table.

Statistical analysis

For all statistical analyses, the results of at least three independent experiments were considered for each case. The differences between KO and WT mice were analyzed using one-way analysis of variance (ANOVA), and differences were considered as significant at p-values < 0.05.

Results

Spats 1 is a highly conserved gene

An updated phylogenetic analysis expanded the conservation degree of SPATS1, revealing its presence in a high number of taxa, and showing that Spats1 originated early in evolution. In particular, blast analyses identified SPATS1 in 202 animal species including cnidarians, mollusks, brachiopods, hemichordates, echinoderms, urochordates, cephalochordates, cartilaginous and bony fish, amphibians, reptiles, and mammals including Monotremes, Metatherians, and all Eutherian orders (Fig 1A and S1 Fig). Curiously, no homologies were found in birds, with the only exception of the order Apterygiformes (i.e. the kiwi from New Zealand), where a predicted protein with a certain homology degree to mouse SPATS1 was identified. Similarly, we have not found homologs in members of the Ecdyzoa clade, including D. melanogaster and C. elegans. The absence of homologies in this clade has been previously reported for other mammalian proteins comprising meiotic proteins such as for instance synaptonemal complex components [33], and has been interpreted to be a consequence of the rapid evolution and sequence diversification of ecdyzoans [33, 34]. No significant similarities were found in plants either. Thus, despite the few above-mentioned exceptions, SPATS1 is highly conserved along the evolution of metazoans. Particularly, the extensive similarity between mouse and human SPATS1 can be seen in Fig 1B (see also S2 Fig for similarities at the nucleotide level).

Fig 1 — (A) Highly resolved tree showing the presence of SPATS1 and its phylogenetic relationship across different metazoans taxa. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. (B) Alignment of SPATS1 amino acidic sequence from mouse (*Mus musculus*) and man (*Homo sapiens*). Alignment was performed with ClustalW and BoxShade. Identical amino acids are highlighted in black and similar amino acids in grey.

In order to evaluate if the expression pattern of Spats1 was also conserved between species, we performed data mining. Expression atlas showed that Spats1 is differentially expressed in the testes compared to other tissues, and this is true for the different analyzed mammalian species, including rat, mouse, man, macaque, horse, bull, sheep, and the marsupial opossum (not shown). Besides, for Spats1 homologues identified beyond mammals and with available information at the NCBI database, a high proportion corresponded to mRNAs isolated from testis. Examples of this are the Spats1 homolog from bony fish Oryzias latipes (GenBank Access number FS557457), cartilaginous fish Callorhinchus mili (access number XP_007901502.1), cephalochordates Branchiostoma belcheri and Branchiostoma floridae (access numbers XP_019647808 and XP_002590276.1, respectively), hemichordate Saccoglossus kowalevskii (access number XP_002731024.1), echinoderm Strongylocentrotus purpuratus (access number XP_001184069.1), brachiopod Lingula anatina (access number XP_013389114.1), and the mollusk Octopus bimaculoides (access number XP_014774497.1), among others. Of note, a recent study in juvenile snakeskin gourami fish also identified Spats1 among the top differentially expressed genes in testis, as compared to ovary [35].

Thus, both the Spats1 gene and its testicular expression pattern appear to be conserved along the evolution of metazoans.

Spats1^-/- mice are fertile and present normal spermatogenesis

The high evolutionary conservation, together with its testis-biased expression pattern, suggested that Spats1 could be an important gene for male reproduction. To investigate this, we generated Spats1^-/- mice by obtaining a 2-bp deletion in exon 3 of the gene, with the use of CRISPR/Cas9 system (Fig 2A and 2B). The deletion resulted in a frameshift at residue 90, to generate a putative aberrant protein of 111 amino acids instead of the WT, 269-residue protein. Western-blot analysis with an antibody raised against the central region of SPATS1 detected the protein in WT animals, but not in Spats1^-/- individuals (Fig 2C).

Fig 2 — (A) Graphical representation of the structure of mouse *Spats1*. Coding exons appear as solid black bars and non-coding exons as white bars. Introns are represented as horizontal lines. Sequence of the sgRNA, designed to target exon 3, is underlined in black, and the PAM sequence is underlined in red. (B) Representative genotyping results obtained through sequencing of PCR products amplified from mouse tail tips. The 2 bp deletion in *Spats1*^-/- mutants is shown. (C) Western blot analysis showing the detection of SPATS1 in testicular lysates of two WT mice, but not in those of 2 KO ones. β-actin was used as a loading control.

The analysis of mutant mice showed no overt defects in Spats1^-/- mice, either male or female. Homozygous mutants of both genders were fertile, and produced offspring of a litter size similar to WT ones (Fig 3A). Besides, no differences in testicular morphology, size or weight, were observed between WT and KO mice (Fig 3B).

In order to have quantitative comparative analyses, testicular cell suspensions from adult WT and Spats1^-/- mice were stained with propidium iodide and analyzed by flow cytometry (FCM), mainly based on DNA content [36]. No significant differences between WT and mutant mice were found, indicating similar relative proportions of either somatic cells and spermatogonia (2C in DNA content), meiotic cells (4C), spermatids (C) (Fig 3C), and spermatozoa (C, apparently sub-haploid left-most peak) (S2 Table) [37].

Histological analysis of young adult mice testes revealed no obvious differences between those of WT and Spats1^-/- animals (Fig 3D). Furthermore, no significant differences in motility per cauda epididymis either for fresh or for capacitated sperm (Fig 3E and 3F), or in epididymal sperm concentration (S3 Fig), were observed among WT and Spats1^-/- mice. Concerning sperm morphology, no significant differences in the proportion of anomalous spermatozoa in KO mice compared to WT ones were observed either (Fig 3G).

It has been reported that in some cases of KO mice no testicular phenotype is observed in young animals, but as the age advances a phenotype starts to develop, such as progressive fertility loss, decreased sperm count [38], or germ cell loss [39]. Therefore, we let some of the animals reach 1 year of age and analyzed their phenotype. Spats1^-/- mice at 1 year of age did not present any evident morphological alteration in comparison to WT, and the males presented testes of normal size and aspect (not shown). Fertility tests showed a slight although not significant difference with their WT littermates (Fig 4A). Histological analysis showed seminiferous tubules with similar appearance to those of WT animals (Fig 4B). Moreover, no significant differences in sperm motility (Fig 4C and 4D) and morphology (Fig 4E) between WT and KO mice were observed. Besides, although some differences in sperm concentration were observed in this case (S3 Fig), again, they were not significant.

Fig 4 — (A) Average litter size, obtained by intercrossing WT individuals, or crossing KO males with WT females. (B) Histological analyses of the testes of WT and *Spats1*^–/–male mice. Scale bar: 25 μm. (C) Total motility (percentage) of sperm from cauda epididymis of WT and *Spats1*^–/–mice, as assessed by CASA. Non-capacitated and capacitated sperm were analyzed. (D) Percentage of sperm with progressive motility, from the cauda epididymis of WT and KO mice. (E) Percentage of sperm from the cauda epididymis with normal head morphology, from WT and KO mice. All the data correspond to the results obtained from the analysis of 3–6 individuals of each type, and are presented as the means ± SD.

No differences in the expression level of genes from the Wnt pathway are detected between the testes of Spats1^-/- and WT mice

An existant report by Zhang et al. [23] suggested that SPATS1 would be a negative regulator of the canonical Wnt pathway. Using transfection of diverse molecular constructions into heterologous somatic cells, in that work it was observed that SPATS1 (therein renamed as DDIP [Dishevelled-DEP domain interacting protein]) was capable of binding and sequestering DVL2 (Dishevelled 2), a positive regulator of the Wnt pathway. According to their results, SPATS1 would weaken the β-catenin/TCF4 interaction by promoting the degradation of transcription factor TCF4 [23]. As spermatogenesis requires a fine-tuning of the Wnt pathway (e.g. [40–42]), we wondered if this pathway would be disregulated in the testes of Spats1^-/- mice. In order to answer this question, we analyzed the expression levels of some of the most common Wnt pathway-target genes that are activated by the β-catenin/TCF4 complex, namely Lef1, Tcf1, Cyclin D1 (Ccnd1), and c-Myc [39, 41, 43–45], in the testes of Spats1^-/- mice, in comparison to WT ones.

The analysis by qRT-PCR showed no significant differences in the expression levels of the target genes of the Wnt pathway in testis between WT and KO mice (except for Lef1, which contrary to what expected, showed lower expression levels in KO mice; Fig 5A). Particularly, c-Myc (for which a role in immature Sertoli cells has been suggested, and whose mRNA is usually not detectable in adult testes [46]) did not show expression above background either in the testes of WT animals or in those of Spats1^-/- ones. Likewise, we did not detect expression of Dvl2 in the testes of either WT or mutant animals. We also analyzed the expression of Dvl1, a Dvl2-related family member with probable functional redundancy [47] that shows stage-dependent expression changes along mouse spermatogenesis [48]. No significant differences in the expression levels of Dvl1 were observed either.

Fig 5 — (A) qRT-PCR of testicular RNA from 55 dpp WT and KO mice. (B) qRT-PCR of testicular RNA from 1 year-old WT and KO mice. Data are expressed as absolute normalized expression levels in arbitrary units (mean ±SD). *Lef1*: *Lymphoid enhancer binding factor 1; Tcf1*: *Transcription factor 1; Ccnd1*: *Cyclin D1; c-Myc*: *Myc proto-oncogene; Dvl2*: *Dishevelled-2; Dvl1*: *Dishevelled-1; Ctnnb1*: *β-catenin; Wnt4*: *Wingless family member 4*.

Wnt4, one of the initiators of Wnt signaling, is a key gene for the development of female genitalia, with its absence leading to partial female-to-male sex reversal in mouse (e.g. [42, 49]). A fine tuning of Wnt4 expression is also implicated in testis development and spermatogenesis [50–55]. Interestingly, in a study Spats1 appeared within the list of upregulated genes in Wnt4-null fetal mouse ovaries, compared to controls [56]. Therefore, here we also evaluated the expression of Wnt4; its mRNA is usually undetectable in normal testes [51], and remained the same in those of KO mice. No differences in the expression levels of the gene coding for the core effector of the pathway, β-catenin (e.g. [25, 40, 42, 57]), were observed either (see Fig 5A).

Progressive spermatogenic defects have been reported for mutant mice with germ cell-specific constitutive activation of β-catenin as age increases [39]. As a consequence, we also analyzed the expression of the above-mentioned downstream targets of the Wnt pathway in the testes of 1 year-old KO mice. However, no significant differences between WT and Spats1^-/- animals were observed (Fig 5B).

Discussion

Some data have allowed us to suspect the importance of SPATS1 for testis development and/or spermatogenesis. First, it shows a testis-restricted expression pattern, and high expression levels in pachytene spermatocytes during the first spermatogenic wave. Second, it has a high evolutionary conservation. As reproduction-related proteins are in general poorly conserved–most probably due to unusually fast evolution rates [58]–this high conservation would suggest an important role for SPATS1. Moreover, a few reports have proposed its link with spermatogenesis, male fertility [20, 21], and testicular cancer [22]. Therefore, and due to the absence of functional studies, we addressed the generation of a Spats1 KO mouse model for the analysis of its functional roles in relation to mouse spermatogenesis and fertility.

As expected for the KO of a testis-specific gene, Spats1^-/- mice were viable, and female individuals were completely normal and fertile. However, unexpectedly, mutant males were fertile as well, and produced offspring of a similar litter size to WT ones. Besides, they showed no differences neither in testicular morphology, size or weight, nor in testicular cell content or histology. In addition, no significant differences in sperm count, morphology or motility between WT and KO mice were observed.

This work adds to an increasing number of studies that show a lack of phenotype for KO mice of testis-specific genes. As an example, Miyata et al. [59] reported the study of KO mice for 54 evolutionarily conserved genes with testis-enriched expression, including some genes believed to be important for fertility. They found that all the KO mutants were fertile, which indicates that those genes were not individually essential for male fertility. A similar result was obtained in another study where KO mice for 30 conserved testis-enriched genes were generated [60]. A possible explanation for the lack of phenotype may be functional redundancy [59, 60]: as reproduction is the “master function”, it is likely that redundant genes to cover a same essential role exist, to ensure that it can be satisfactorily fulfilled. This could be the case for some reproductive genes where related family members may compensate for the KO gene-loss of function (e.g. [61–64]). Although SPATS1 would not be a member of a protein family, we cannot rule out the possibility of other proteins with overlapping function.

Sometimes reproduction-linked phenotypes of KO mice may become evident with age [38, 39]. However, this does not seem to be the case for Spats1^-/- animals, as we failed to detect differences with WT ones in 1 year-old individuals.

On the other hand, it must be recalled that reproduction is extremely sensitive to the environment, and therefore some reproduction-related genes may not be essential in normal laboratory conditions but be required under other, more stressful circumstances. Moreover, disruption of a single gene may not cause an evident effect on fertility, but it may have dramatic consequences in combination with environmental factors, or with other mutations or polymorphisms [8, 65]. If this were the case for Spats1, it could possibly reconcile the discrepancies between the lack of phenotype of Spats1 KO mice, and the reports that associate alterations or polymorphisms of Spats1 with human male pathologies such as infertility and testicular cancer [20, 22]. Furthermore, we cannot exclude the possibility that the absence of SPATS1 be better tolerated in mice than in humans.

Finally, a report using transfections in heterologous cell culture suggested that SPATS1 would be a negative regulator of the Wnt pathway [23]. We considered this an attractive idea, as accumulating evidence indicates the relevance of Wnt signaling in testis development and physiology, and its alteration in different male reproductive anomalies (e.g. [40, 42, 66]). However, when comparing Spats1 KO vs WT mice, we could not find any significant change in the expression of common Wnt pathway-target genes in the testis, which is the tissue where Spats1 is mostly expressed. In this regard, we remark the relative validity of heterologous somatic cell cultures and transpolation of results to the complex testicular tissue, and the obvious importance of performing in vivo studies [67], such as the generation of loss-of-function mutants. Thus, although of course we cannot rule out the possibility of a relation between SPATS1 and the Wnt pathway, we must state that in this study we did not find any evidence of this relation in testis.

In summary, this study adds Spats1 to the list of evolutionarily conserved genes with a testis-restricted/differential expression pattern that are individually dispensable for mouse spermatogenesis and fertility. While according to other reports a defect in Spats1 may be a risk factor for male testicular health in human and bull [20–22], we here show that the lack of SPATS1 protein alone does not cause an evident phenotype in mouse. This information is important for the scientific community in order to avoid duplicate efforts from other laboratories. Besides, it is useful in the search for potential male contraception targets, as it discards Spats1 as a potential target.

Supporting information

S1 Table. Primers used in this study.

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Click here for additional data file.^{(187.7KB, pdf)}

S2 Table. Quantification of the different cell populations in testicular cell suspensions from WT, Spats1^+/- and Spats1^-/- mice, by FCM.

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Click here for additional data file.^{(18.7KB, pdf)}

S1 Fig. BLAST alignment of SPATS1 homologous sequences from different metazoan species.

Mus musculus protein sequence was used as query. Alignment was performed with ClustalW, and visualized using Jalview 2.11.1.4 (https://mybiosoftware.com/jalview-2-6-1-multiple-alignment-editor.html). The most conserved positions along evolution (from 0 to 9) are shown below.

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Click here for additional data file.^{(5.7MB, tif)}

S2 Fig. Alignment of Spats1 coding sequences from mouse (Mus musculus) and man (Homo sapiens).

Alignment was performed with ClustalW and BoxShade.

(TIF)

Click here for additional data file.^{(620KB, tif)}

S3 Fig. Relative sperm count (millions/mL) from cauda epididymis of WT and KO mice.

(A) 45–60 dpp animals. (B) One year old individuals. Although the count is not completely reliable in absolute numbers (as obtained through swimming out from the cauda), the data is comparable between samples.

(TIF)

Click here for additional data file.^{(917.5KB, tif)}

S1 Raw images

(PDF)

Click here for additional data file.^{(1.5MB, pdf)}

Acknowledgments

We thank BSc Gabriel Fernández-Graña (UATE, IPMontevideo) for animal care and crossbreeding. We are grateful to Dr Rossana Sapiro (Histology Department, Faculty of Medicine, UdelaR) for generously allowing us to use the CASA equipment, and for her help with it. MSc Federico Santiñaque is also thanked for his excellent technical assistance with FCM.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by Comisión Sectorial de Investigación Científica (CSIC), UDELAR, Uruguay (https://www.csic.edu.uy/), under an I+D Groups 2018 grant to AG and RB. CAC was awarded with a PhD scholarship from Agencia Nacional de Investigación e Innovación (ANII), Uruguay (https://www.anii.org.uy/). Experiments carried out at UATE, IPMontevideo, were supported by FOCEM (MERCOSUR Structural Convergence Fund; https://focem.mercosur.int/es/), COF 03/11. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0251028.r001

Decision Letter 0

Suresh Yenugu

15 Mar 2021

PONE-D-21-03796

SPATS1 (Spermatogenesis-associated, serine-rich 1) is not essential for spermatogenesis and fertility in mouse

PLOS ONE

Dear Dr. Geisinger,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please include additional information on the functional role of SPATS in relation to evolutionary process. The functional relevance of the genes analysed of a particular pathway be elaborated in the discussion. Reanalyze the sperm count with tissue homogenates and using stage specific sections for histological anlyses are recommended.

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Reviewer #1: In this manuscript, Capoano and colleagues report the generation of a KO mouse model for the gene SPATS1. This Serine-rich gene is the member of a 3 gene-family (including SPATS1, 2 and L2) and is found in many animals. Its expression has been reported in the testis either prior or during meiosis.

The authors used CRISPR technology to induce a frame-shifting mutation in the 3rd exon of SPATS1. The manuscript investigates the potential defects in reproductive fitness of KO animals and finds no major alteration in germ cell development or mating success. Thus, the author conclude that it is not essential for fertility in mouse.

The work is original, as no other SPATS1 KO mouse models have been published to my knowledge. The research is presented clearly and appropriately. Overall, this manuscript meets all the standard of research ethics and conclusions are supported by the data - with the few clarifications needed and described below.

In the following section, I provide revision points which should clarify and strengthen the manuscript.

1) In the abstract (i.e. last sentence) and throughout the text the authors should be careful about making parallels between Human fertility phenotypes related to SPATS1 and the mouse phenotypes described here. It is very possible – as the authors briefly note in the discussion – that one is not a good model for the other, as it is the case with many other spermatogenesis genes.

Please also carefully consider the next comments regarding SPATS1 evolution in the following points.

2) Page 4 Line 78-80: What is the proposed function across evolution? Meiosis? Is it a sex-specific gene other metazoan?

3) Page 12 Line 280-281: Clarify this sentence. What is the observation?

4) The conclusion from figure 1 need to be developed to fully support the authors conclusions: the authors need to show an alignment or a few representative sequences to support the claim that it is “highly conserved” (again to distinguish between evolutionarily retained and conserved at the sequence level). Alternatively, they could use branch length on the tree to support this claim. Finally, the authors need to specify what type of tree is displayed and the scale used (substitutions per site maybe?) in Fig1.

5) Page 13 line 299: What does differentially expressed means here? Relative to what? Please clarify.

6) Can the author specify how many backcrosses in which strain background was made to produce the experimental set of mice?

7) Can the author specify where the antibody maps relative to the full-length protein and frame-shifted (truncated) sequence?

8) Page 17 Line 412-415: maybe the author could show results of these qPCRs for 4C cells.

9) Page 17 Line 419: please indicate here what is the expected level and function of c-Myc in WT testes.

10) Page 21 Line 483: I would not be as categorical that SPATS1 is *not* a member of a multi-gene family. There is also SPATS2 and 2L in the mouse and human genome. Not to mention the SPATA gene family…

Reviewer #2: Thank you for the opportunity to review this article. The authors generated a knockout Spats1 mouse model to investigate the role of SPATS1 in male fertility. Knockout male and female mice were fertile and showed no signs of any reproductive defects. While the paper is mostly rounded, I provide a few comments below and suggestions that I believe will improve the robustness of the article. I recommend that the article is accepted conditionally to these being completed.

Comments:

Line 32 – SPATS1 loss of function or overexpression? – I note this is further fleshed out well below in lines 81-91

Line 37 – does ‘structure’ = histology?

Line 58 – peculiarities? Probably should just delete this word

Line 130 – “testes were weighed individual after removal of the tunica?” The tunica does not need to be removed and probably would cause more artefacts than when testes are kept intact. Why was this approach used?

Line 163 – were the males also 45 days of age?

Line 170-172 – you could just state Spats1 KO or WT were mated with …

How long were they mated for?

Line 187 – is this a hybrid between EM and basic light histology approaches?

Line 228 – how were sperm recovered? By cutting the cauda or by retrograde perfusion?

Line 246 – typo: “autors”

Results:

Thank you to the authors for ageing the mice to 1 year to determine if there were any age-related phenotypes, this is a good approach.

Figure 3A – small suggestion – change the polka dot legend to show male first like in all the others

B – change the axis to g instead of gr – you could label these as Bi, Bii, Biii

D – these are quite beautiful and especially the lower right cross-section. It is clear that spermatogenesis looks normal, however the tubules on lower left and right and not stage matched. If you could change the WT to match the KO or vice versa that would be more appropriate. It is much simpler probably to just do normal 5 um sections and PAS stain the testes however for a similar result.

E- I do not think it was stated how concentration was measured but depending on the method used to extract sperm (if the caudae were simply nicked) then counting the resulting sperm is not accurate. The error bars are large so it might be useful to omit this panel. Truly accurate counts can only be done with homogenisation of the epididymis or the cauda specifically, as other methods do not fully recover the sperm from the epididymides.

F – progressive motility looks fine, however, the authors could also show the total motility and replace the panel to the left with this

Line 355 – no significance and it looks very similar for head defects, so it is best to just state no difference

Fig 4A – the legend could go underneath the columns

C – comment as above

B – The tubules are not stage-matched and are decent but not as good quality as in the above figure.

Discussion:

The discussion covers all topics of the m/s. Line 455 – a few hints – could be rephrased

**********

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PLoS One. 2021 May 4;16(5):e0251028. doi: 10.1371/journal.pone.0251028.r002

Author response to Decision Letter 0

5 Apr 2021

Dear Editor,

Thank you for considering our manuscript “SPATS1 (Spermatogenesis-associated, serine-rich 1) is not essential for spermatogenesis and fertility in mouse” ([PONE-D-21-03796] - [EMID:45ee38e7b541001a]) for publication in PLOS ONE. We thank all the comments and suggestions, that helped improve the ms. We are now submitting the corrected version. All the received comments from the reviewers have been considered, and we are hereby responding to all of them in a detailed fashion. Most of the suggested corrections have been included in the revised ms.

Besides, following the journal requirements, we have provided the original uncropped and unadjusted Western blot image (S1_raw images), and amended the ethics statement to include the full name of the ethics committee that approved our specific study (now Lines 122-123 and 130-131 of the revised version with track changes).

Editor:

Reply: Thanks for the suggestions; they have been incorporated.

Specifically, as indicated, SPATS1 is evolutionarily conserved along metazoans. Now, following the recommendations from Reviewer 1, we have included the alignment of SPATS1 from mouse with that of human, and also with the predicted SPATS1 sequences from several species, from invertebrates to mammals. As stated in the Discussion, reproduction-related proteins are in general poorly conserved (most probably due to unusually fast evolution rates), and therefore this high conservation would suggest an important, conserved role for SPATS1. However, despite its high evolutionary conservation, the functional role of SPATS1 is unknown. Moreover, as shown in this ms, it is not individually essential for male fertility.

Concerning the reanalysis of sperm count, we re-analyzed the sperm count in testicular cell suspensions by flow cytometry, which represents a widely accepted means to analyze testicular cellular content, with very high quantitative analytical power and statistical weight (now S2 Table). We also replaced the histological images with matched stages as suggested. A detailed point-by-point description is in the response to reviewers below.

Reviewer #1:

Reply: Done. Thank you for the comment. We have now clarified that SPATS1 is not essential for male fertility in mouse (see lines 45 and 95 of the revised version with track changes). Besides, although previously stated, we further emphasized that the studies that had suggested a link with male testicular health were from human and bull (line 539).

The gene is present, that seems clear. Yet, considering the scope of the study, I would recommend showing an alignment of mouse and human SPATS1 sequences (CDS and Proteins) to point to the regions that are indeed “conserved”. Please also carefully consider the next comments regarding SPATS1 evolution in the following points.

Reply: Done. Following the reviewer´s suggestion, in the revised version we have included an alignment of mouse and human SPATS1 sequences, both for proteins (newly included Fig 1B) and for CDS (newly included S2 Fig), to show the extensive similarities. We have also payed careful attention to the related comments addressed in the following points (see below).

2) Page 4 Line 78-80: What is the proposed function across evolution? Meiosis? Is it a sex-specific gene other metazoan?

Reply: There is no clue about the possible function. Yes, it is a testis-specific gene in every species for which we could find expression-related information in databases. The reviewer is right that “supports the idea that this protein could have an important, conserved role in testis” can be interpreted as if an important role for this protein had been proposed. As the idea is based on the high conservation level (in this regard, it must be recalled that reproductive proteins are in generally poorly conserved, as stated in the Discussion section), we have now replaced “supports the idea” with “allows to suspect” (line 79).

3) Page 12 Line 280-281: Clarify this sentence. What is the observation?

Reply: Done. The wording of the sentence has been changed to clarify the sentence. We hope it is clearer now (now lines 286-290).

Reply: Done. Thank you for the suggestion. We have now included an alignment of about 170 species (newly added S1 Fig). This alignment includes most of the species that were used to make the tree from Fig 1A. Besides, in the legend to Fig 1A we have explained that the tree is indeed a substitutions per site tree (it is represented to scale, with branch lengths representing the number of substitutions per site; lines 303-304), and the scale is shown in the figure. Sorry for not clarifying it before.

5) Page 13 line 299: What does differentially expressed means here? Relative to what? Please clarify.

Reply: Sorry, the reviewer is right. Spats1 is differentially expressed in the testis compared to other tissues. The sentence has been now reformulated to clarify this (now line 312).

6) Can the author specify how many backcrosses in which strain background was made to produce the experimental set of mice?

Reply: Three backcrosses to C57BL/6J were done. This information has been now included in the text (lines 151-152).

7) Can the author specify where the antibody maps relative to the full-length protein and frame-shifted (truncated) sequence?

Reply: This was a commercial antibody, and the exact amino acidic sequence was proprietary information from the company. Anyway, by asking the company we could find out that it spanned the central region of the protein (as stated in the manuscript), around amino acid 186. As the frameshift in the KO protein is at aa 90 (thus missing the central and C-t regions, which are the most highly conserved along evolution) this antibody would not recognize the mutant protein.

8) Page 17 Line 412-415: maybe the author could show results of these qPCRs for 4C cells.

Reply: Thank you for the suggestion. In fact, we are not sure whether the targets of the Wnt pathway should be mostly expressed in 4C cells. Although the highest SPATS1 expression levels were detected in spermatocytes (and even more so during the first spermatogenic wave), relatively high expression levels were also detected in other testicular cell types, including Sertoli cells, spermatogonia, and peritubular myoid cells. Furthermore, in embryonic testes SPATS1 is detected in gonocytes (at least in the rat).

The targets of the Wnt pathway have been detected in different testicular cell types as well. Most reports about testicular disruption of the Wnt pathway show overexpression of Wnt targets in Sertoli cells and spermatogonia (e.g. in mutants with altered β-catenin expression). On one hand, aberrant activation of Wnt signaling targets in testis disrupts Sertoli cells differentiation and their ability to support spermatogenesis, resulting in seminiferous tubule degeneration and infertility. However, the different reports are contradictory concerning the developmental moment in which this effect is observed, ranging from embryonic life to adulthood (e.g. Boyer et al., 2008, doi 10.1095/biolreprod.108.068627; Chang et al., 2008, doi 10.1242/dev.018572; Tanwar et al., 2010, doi 10.1095/biolreprod.109.079335). On the other hand, increasing evidence suggests that both disruption and overexpression of the Wnt pathway specifically in male germline also affects spermatogenesis. For instance, a study in which the Wnt pathway was overactivated in the germ line through constitutive β-catenin expression, showed overexpression of the downstream targets (TCF1, Lef1, cyclin D1) in germ cells, and particularly in spermatogonia (Kumar et al., 2016, doi 10.18632/oncotarget.13920). Again, there are inconsistencies concerning the exact timing (e.g. Chang et al., 2011, doi:10.1371/journal.pone.0028039; Kerr et al., 2014, doi: 10.1095/biolreprod.112.105809; Kumar et al., 2016, doi 10.18632/oncotarget.13920; Chassot et al., 2017, doi: 10.1016/j.ydbio.2017.04.010). In addition, discrepancies between in vitro and in vivo results are not uncommon (e.g. Xue et al 2021, doi 10.1093/molehr/gaaa085).

As a consequence, due to the intricate signaling within the seminiferous epithelium (e.g. between somatic and Sertoli cells), and to the complexity of Wnt signaling itself, in case the Wnt pathway would be affected by the absence of SPATS1, we cannot be sure if we should expect the effect particularly in spermatocytes or in other cell types as well. This is the reason why we decided to analyze it in whole testes.

This being said, we agree that it could be interesting to analyze the expression of Wnt targets in isolated cell types (i.e. 4C cell population, and maybe also 2C), to see if at least some subtle expression differences were detected in specific cell types. Unfortunately, due to Covid 19 pandemic, the transgenic facility significantly reduced its staff and workload, discontinuing most of the lines (including Spats1 KO) and freezing embryos. We must also take into consideration that in testicular studies in which some significant alterations of the expression levels of the Wnt pathway targets were observed, these were usually acccompanied by dramatic phenotypic changes (alterations in testis development, infertility, and/or testicular cancer [e.g Kerr et al., 2014; Kumar et al., 2016; Lanza et al., 2016, doi: 10.1080/15384101.2016; Chassot et al., 2017]). Therefore, as Spats1 KO mice do not present an overt phenotype, we think that the effort of reviving the mouse line to repeat the qRT-PCR assays in isolated cell populations is not worth it, as it is highly unlikely that even subtle changes in gene expression will be detected.

9) Page 17 Line 419: please indicate here what is the expected level and function of c-Myc in WT testes.

Reply: Done. We have now indicated it in the text of the revised version (now lines 439-440).

Briefly, the transcription factor/proto-oncogene c-Myc is one of the most common targets of the Wnt pathway that are activated by the β-catenin/TCF4 complex (He et al., 1998, doi 10.1126/science.281.5382.1509), together with Lef1, Tcf1, cyclin D1.

Particularly in WT testis, c-Myc has been shown to be expressed in Sertoli cells from 8-day-old rats but hardly detectable in cells from those aged 14 and 28 days, and a role in immature Sertoli cells has been suggested (Meroni et al 2019, doi: 10.3389/fendo.2019.00224). Interestingly, different reports have detected c-Myc overexpression in adult testes of mice and rats under abnormal conditions such as treatment with toxic substances (e.g. Wang et al., 2020, doi 10.1111/and.13841; Somade et al., 2020, doi: 10.1016/j.metop.2020.100051).

In summary, c-Myc expression is not expected to be observed in WT adult testis. As the study referred in the manuscript (using heterologous cell culture transfections) suggested that SPATS1 would be a TCF4 repressor, we aimed at finding out whether c-Myc (as well as other common targets) was upregulated in Spats1 KO mice. Furthermore, since – as mentioned in the ms – a report suggested a possible link between Spats1 mutation and human testicular cancer, we found it interesting to analyze if the expression levels of c-Myc (a proto-oncogene) were altered in the testes of Spats1-deficient mice.

Reply: Thank you for the comment. To the best of our knowledge, SPATS2 and SPATS2L do not have any sequence similarity to SPATS1. BLAST alignments indicate that: “No significant similarity was found”, neither in mouse nor in human, and neither at the protein nor at the nucleotide levels (SPATS2 and 2L have partial similarity between them, though). Besides, according to our analyses, they do not share any common domain or other common trait.

In the original paper where SPATS2 was described, and named p59scr (Senoo et al., Biochem Biophys Res Commun 292:992, 2002; doi: 10.1006/bbrc.2002.6769), the authors state: “The entire protein was rich in serine residues (13.6%), especially within amino acids between 373 and 411 (35.0%). Because of these characteristics, we named this novel protein p59scr (59 kDa, Serine-rich Spermatocytes and Round spermatid protein).” It is unfortunate that due to its overall serine content and testicular expression pattern, GenBank gave it a similar name to SPATS1, which may lead to confusion.

The ClustalW alignment for Mus musculus protein sequence between SPATS1 and SPATS2, below, shows the lack of similarity:

Spats1 ------------------------------------------------MESSKDTQHGDA

Spats2 MSRKQSQKDSSGFIFDLQSNTVLAQGGTFENMKEKINAVRAIVPNKSNNEIILVLQHFDN

* ** *

Spats1 LESKSCLANRTSSR------------------QNKRTSLSSSDGTGPRVTESLGLPRVLT

Spats2 CVDKTVQAFMEGSASEVLKEWIVTGKKKNKKKKSKPKPASEASGSAPDSSKSAPIQEEQP

.*: * .* :.* .. *.:.*:.* ::* : . .

Spats1 PSDTAAELG---------------------------------------------------

Spats2 ASSEKGSINGYHVNGAINDAESVDSLSEGLETLSIDARELEDPEFAAAETLDRTGSVLEN

.*. ..:.

Spats1 ------------------QKTSSSSSSSSSSAQSNRSSKVSLPEIPK-------------

Spats2 GVSDFEPKSLTAHSISNVQQSRNAAKSLSRTTPGAQVSNLGMENVPLSSTNKKLGSNIEK

*:: .::.* * :: . : *::.: ::*

Spats1 ---------------------------EKYPEEFSLLNSQTEDGQRPEWTFYPRFSSNIH

Spats2 SVKDLQRCTVSLARYRVVVKEEMDASIKKMKQAFAELQSCLMDREVALLAEMDKVKAEAM

:* : *: *:* * : . : :..::

Spats1 TYHIG------------------KQCFFNGVFRGNRRSVAERTVDNSLGKKKYDIDPRNG

Spats2 EILLSRQKKAELLKKMTDVAVRMSEEQLVELRADIKHFVSERKYDEDLGRVARFTCDVET

:. .: : : . :: *:**. *:.**: :

Spats1 IPKLTPGDNPYMFPEQSKEFFKAGATLPPVNFSLGPYEKKFDTFIPLEPLPKIP------

Spats2 LKQSIDSFGQVSHPKNSYSTRSRCSLVAPVSLSG-PSDGSAASSSPDASVPSLPGANKRN

: : . . .*::* . . : :.**.:* * : . : * .:*.:*

Spats1 NLPFWEKEKANN----------------------LKNEIKEVEELDN-------------

Spats2 CAPREASAAMTNSSDRPCQAHREVFPGNRRGGQGYRAQSQKTADPSNPGRHDSVGRYRNS

* . .* : : ::. : .*

Spats1 -WQVPMPFLHGFFSTGASNFSRQQ------------------------------------

Spats2 SWYSSGPRYQGVPPQAPGNAGERSRPYSAGTNGTGAISEPSPPKPSFKKGLPQRKPRASQ

* . * :*. . ...* ..:.

Spats1 ------

Spats2 AEAANS

Regarding the Spata (spermatogenesis-associated) family, despite that one of the synonymous names that appear in GenBank for human (but not for mouse) SPATS1 is SPATA 8, Spats1 is unrelated to the rest of Spata genes. Moreover, a tblastn search among all GenBank conducted with mouse SPATS1 sequence and excluding Spats1 (in order to find out if there were possible matches with other sequences but Spats1 homologous sequences) gives the message “Not significant similarity was found”, not producing any matches with SPATS2, the products of any Spata gene, nor any other predicted proteins in GenBank database.

Reviewer #2:

Line 32 – SPATS1 loss of function or overexpression? – I note this is further fleshed out well below in lines 81-91

Reply: Done. We have now clarified this in the text (line 33).

Line 37 – does ‘structure’ = histology?

Reply: Thanks for the observation. It has been amended by replacing “structure” by “histology” (now line 38).

Line 58 – peculiarities? Probably should just delete this word

Reply: Done. It has been deleted.

Reply: The reviewer is right that removal of the tunica albuginea may cause some distortion. However, we are used to removing the tunica (which we do very cleanly), because we usually employ the tissue for preparing testicular cell suspensions. Besides, we wanted to get an idea of the weight of the testicular parenchyma.

Line 163 – were the males also 45 days of age?

Reply: Yes, they were also 45 days of age. It has been now clarified (now line 167).

Line 170-172 – you could just state Spats1 KO or WT were mated with …

How long were they mated for?

Reply: Done. The sentence was reformulated according to the reviewer´s suggestion, and the mating time (three months) was included (now lines 174-175).

Line 187 – is this a hybrid between EM and basic light histology approaches?

Reply: We usually prefer this method for sample preparation for microscopy because it involves a double fixation, first with glutaraldehyde and then with osmium tetroxide, thus enabling an excellent preservation of testicular histology. Besides, although in this case semi-thin sections and optical microscopy were used, the same samples can be eventually employed for thin sections in case transmission electron microscopy (TEM) would be required.

Line 228 – how were sperm recovered? By cutting the cauda or by retrograde perfusion?

Reply: Sperm were recovered by cutting the cauda. We have now explained it in the text (now line 234).

Line 246 – typo: “autors”

Reply: Sorry, thank you for noting it. We have corrected it (now line 252).

Figure 3A – small suggestion – change the polka dot legend to show male first like in all the others

Reply: Done, thank you for the suggestion.

B – change the axis to g instead of gr – you could label these as Bi, Bii, Biii

Reply: Done. gr has been replaced by g, and the different parts have been labeled as suggested.

Reply: Done. Thanks for the suggestion. We have replaced the lower left image (WT), to match stage with the lower right (KO).

Reply: A fixed number of nicks were performed in each cauda (it is now explained in Materials and Methods, line 234). Following the reviewer´s recommendation, in the revised version we have omitted this panel from Fig 3E, and from Fig 4C as well.

We agree that this method is not accurate as it does not fully recover the sperm. However, we have made exactly the same cuts in each cauda, and let the sperm swim out for exactly the same time lapse (15 min) in the same conditions and in exactly the same volume, and it was performed by the same operator. Therefore, we think that - although not accurate in absolute numbers - the data is comparable between samples. As it can be useful to show the absence of significant differences between WT and KO (the error bars are large because there were individual differences, but not between WT and KO), we included it now as a supplementary figure (S3 Fig). However, we can still remove it if the reviewer disagrees.

Besides, we re-analyzed the sperm count in testicular cell suspensions by flow cytometry, which, as stated above, represents a widely accepted means to analyze testicular cellular content, with very high quantitative analytical power and statistical weight (now S2 Table).

F – progressive motility looks fine, however, the authors could also show the total motility and replace the panel to the left with this

Reply: Done. We have replaced Fig 3E with a panel showing total motility.

Line 355 – no significance and it looks very similar for head defects, so it is best to just state no difference

Reply: Thanks for the suggestion. We have modified it according to the reviewer´s suggestion, indicating that no significant differences were found (now lines 368-370).

Fig 4A – the legend could go underneath the columns

Reply: Thank you for the suggestion. We tried placing the legend underneath the columns, but we got the impression that it did not look good, as it was too long (because it indicates a cross). Besides, the style is standardized with that in Fig 3A that also shows the results of fertility tests.

C – comment as above

Reply: Done. Following the reviewer´s recommendation, we have eliminated Fig 4C, and replaced it with a panel showing total motility.

B – The tubules are not stage-matched and are decent but not as good quality as in the above figure.

Reply: Done. Thank you for the suggestion. We have changed the images and replaced them with stage-matched tubules.

Discussion:

The discussion covers all topics of the m/s. Line 455 – a few hints – could be rephrased

Reply: Done. We have rephrased the sentence, by replacing “A few hints” with “Some data have allowed us to suspect…” (now line 477).

We hope that with these corrections and clarifications, the ms will now be suitable for publication in PLOS ONE. In case there would be any additional corrections or suggestions, please let us know.

Sincerely,

Adriana Geisinger

Corresponding autor

Attachment

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PLoS One. doi: 10.1371/journal.pone.0251028.r003

Decision Letter 1

Suresh Yenugu

19 Apr 2021

SPATS1 (Spermatogenesis-associated, serine-rich 1) is not essential for spermatogenesis and fertility in mouse

PONE-D-21-03796R1

Dear Dr. Geisinger,

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Reviewer #1: All comments have been addressed

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**********

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Reviewer #1: Yes

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**********

3. Has the statistical analysis been performed appropriately and rigorously?

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**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

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**********

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**********

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Reviewer #1: In their responses to the reviewers, the authors have carefully addressed all of the comments.

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Reviewer #2: Thank you for your response to all my questions and comments. I think the manuscript now stands in an improved state and is ready for publication.

**********

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PLoS One. doi: 10.1371/journal.pone.0251028.r004

Acceptance letter

Suresh Yenugu

21 Apr 2021

PONE-D-21-03796R1

SPATS1 (Spermatogenesis-associated, serine-rich 1)is not essential for spermatogenesis and fertility in mouse

Dear Dr. Geisinger:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Primers used in this study.

(PDF)

Click here for additional data file.^{(187.7KB, pdf)}

S2 Table. Quantification of the different cell populations in testicular cell suspensions from WT, Spats1^+/- and Spats1^-/- mice, by FCM.

(PDF)

Click here for additional data file.^{(18.7KB, pdf)}

S1 Fig. BLAST alignment of SPATS1 homologous sequences from different metazoan species.

(TIF)

Click here for additional data file.^{(5.7MB, tif)}

S2 Fig. Alignment of Spats1 coding sequences from mouse (Mus musculus) and man (Homo sapiens).

Alignment was performed with ClustalW and BoxShade.

(TIF)

Click here for additional data file.^{(620KB, tif)}

S3 Fig. Relative sperm count (millions/mL) from cauda epididymis of WT and KO mice.

(TIF)

Click here for additional data file.^{(917.5KB, tif)}

S1 Raw images

(PDF)

Click here for additional data file.^{(1.5MB, pdf)}

Attachment

Submitted filename: RESPONSE TO REVIEWERS.docx

Click here for additional data file.^{(247.6KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

SPATS1 (spermatogenesis-associated, serine-rich 1) is not essential for spermatogenesis and fertility in mouse

Carlos A Capoano

Luis Adrián Ortiz-Laquintana

Rosana Rodríguez-Casuriaga

Geraldine Schlapp

María Noel Meikle

Ana Paula Mulet

Martina Crispo

Ricardo Benavente

Adriana Geisinger

Roles

Abstract

Introduction

Materials and methods

Evolutionary conservation

Animals

Production of CRISPR/Cas9 knockout mice

Genotyping

Fertility test

Western blot

Histological analysis

Analysis of testicular cell populations by flow cytometry (FCM)

Sperm motility and morphology

qRT-PCR

Statistical analysis

Results

Spats 1 is a highly conserved gene

Fig 1. Conservation of SPATS1 in metazoans.

Spats1-/- mice are fertile and present normal spermatogenesis

Fig 2. Genomic structure and knockout strategy of Spats1.

Fig 3. Fertility and spermatogenesis analyses of Spats1-/- young adult mice of 45–60 dpp.

Fig 4. Fertility and spermatogenesis analyses of Spats1-/- 1 year old mice.

No differences in the expression level of genes from the Wnt pathway are detected between the testes of Spats1-/- and WT mice

Fig 5. Comparative expression analysis of genes of the Wnt pathway in Spats1-/- and WT mice.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Suresh Yenugu

Roles

Author response to Decision Letter 0

Decision Letter 1

Suresh Yenugu

Roles

Acceptance letter

Suresh Yenugu

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Spats1^-/- mice are fertile and present normal spermatogenesis

Fig 3. Fertility and spermatogenesis analyses of Spats1^-/- young adult mice of 45–60 dpp.

Fig 4. Fertility and spermatogenesis analyses of Spats1^-/- 1 year old mice.

No differences in the expression level of genes from the Wnt pathway are detected between the testes of Spats1^-/- and WT mice

Fig 5. Comparative expression analysis of genes of the Wnt pathway in Spats1^-/- and WT mice.