Ybx1 deficiency impairs spermatid development and male fertility without affecting meiosis in mice: insights into spermatogenesis

Abstract

Spermatogenesis is a complex process that is required for sperm production. Multiple RNA-binding proteins participate in regulating spermatogenesis. Y-box-binding protein 1 (YBX1) is involved in transcriptional regulation, mRNA stabilization, and translational repression. However, its specific role in spermatogenesis remains unclear. This study investigated the role of YBX1 in spermatogenesis using a Ybx1 conditional knockout (Ybx1 cKO) mouse model. By analyzing the phenotype of Ybx1 cKO mice, we investigated the role of YBX1 in spermatogenesis and male fertility. The morphology and weight of Ybx1 cKO mouse testes were similar to those of wild-type (WT) testes. Sperm count and motility were lower in Ybx1 cKO mice than in WT mice. Histological analysis showed reduced numbers of elongated spermatids in seminiferous tubules and spermatozoa in tubules of the epididymis in Ybx1 cKO mice. Although YBX1 was highly expressed in the cytoplasm of spermatocytes, meiosis progressed normally in Ybx1 cKO spermatocytes. Finally, the fertilization potential of spermatozoa from Ybx1 cKO epididymis was decreased. In conclusion, our results indicate that YBX1 participates in the regulation of spermatid development but is dispensable for meiosis.

Nucleic acid-binding proteins play a vital role in cellular functions by participating in the regulation of transcription and translation as well as in mRNA processing and stabilization. To date, several RNA-binding proteins, such as BRCA2 [1] and SYMPK [2] for mRNA splicing, RBM46/YTHDC2/MEIOC for post-transcriptional regulation [3], and FXR1 for activating the translation of stored mRNAs, have been reported to be required for spermatogenesis in the germ cells of mammalian testes [4].

Y-box binding proteins are well-characterized DNA/RNA-binding proteins that contain a cold-shock domain for DNA/RNA binding. It was previously reported that Y-box binding proteins are involved in RNA-associated processes, including the formation of messenger ribonucleoprotein complexes (mRNPs), mRNA translation, and stabilization [5]. Y-box-binding protein 1 (YBX1) and YBX2 are notable proteins. In the testes, YBX2 is expressed in meiotic and postmeiotic germ cells and potentially functions in mRNA storage and stabilization. YBX2-binding mRNA is stored in the cytoplasm of male germ cells and required for male gamete development [6, 7]. To stimulate the translation of mRNA stored by YBX2, PAIP1, a protein translation enhancer, is coexpressed and interacts directly with YBX2 [8]. YBX2 knockout leads to spermatogenic arrest through increased mRNA instability [7]. However, its function of YBX1 in spermatogenesis remains unclear.

YBX1, a well-studied Y-box-binding protein, has been implicated in a wide range of human diseases. In brown adipose tissue, YBX1 overexpression promotes brown adipogenesis and thermogenesis through the post-transcriptional regulation of PINK1/PRKN-mediated mitophagy [9]. In renal cell carcinoma, YBX1 promotes renal cell carcinoma cell metastasis by interacting with G3BP1 [10]. In triple-negative breast cancer, YBX1 activates PARP1-related repair by interacting with MEIOB [11], which is a critical protein involved in homologous recombination during meiosis [12]. In zebrafish, maternal YBX1 plays a crucial role in facilitating oocyte maturation and maternal-to-zygotic transition by inhibiting global translation [13]. YBX1 also plays an essential role in implantation development because Ybx1 deficiency leads to embryonic lethality at E13.5 [14]. In early porcine embryos, Ybx1 knockdown led to endoplasmic reticulum stress and increased autophagy and apoptosis [15]. During preimplantation development, the deletion of Ybx1 arrests embryo development at the two- and four-cell stages through the regulation of alternative splicing and maternal mRNA decay [16]. One study that compared proteomic data from the testicular tissues of patients with impaired and normal spermatogenesis revealed decreased expression of YBX1 in the impaired spermatogenesis group [17]. During spermatogenesis, YBX1 interacts directly with the m6A reader PRRC2A, which is essential for completion of meiosis I [18]. These studies indicate that YBX1 is a multifunctional protein that plays vital roles in spermatogenesis.

Although several studies have determined the role of YBX1 in cancer and embryonic development, its function of YBX1 in spermatogenesis remains unknown. This study aimed to investigate the role of YBX1 in spermatogenesis using a Ybx1 conditional knockout (cKO) mouse model. Although highly expressed in the cytoplasmic compartment of spermatocytes, normal meiosis progressed in Ybx1 cKO mice. The fertility of Ybx1 cKO male mice was reduced by a decline in sperm quality.

Materials and Methods

Animals

All the mice were housed at the Experimental Animal Center of Wuhan University. They were housed in a specific pathogen-free (SPF) facility with a 12-h light/dark cycle, maintaining a room temperature of 22 ± 2°C. Standard diet and clean filtered water were provided. All procedures involving animal experiments were approved by the Institutional Animal Care and Use Committee of Wuhan University and conducted in compliance with animal protocols. Mice with loxP sites flanking exons 4–6 of the Ybx1 gene were kindly provided by Professor Haojian Zhang of Wuhan University. Stra8-Cre transgenic mice were provided by Professor Minghan Tong of the State Key Laboratory of Molecular Biology, Shanghai. To generate Ybx1^f/- and Stra8-Cre mice (referred to as Ybx1 cKO), Ybx1^f/f female mice were mated with Ybx1^f/+ Stra8-Cre males. The genotypes of the animals were determined by tail DNA extraction, followed by polymerase chain reaction (PCR) using the designated primers (Supplementary Table 1). The mice were sacrificed by cervical dislocation. The animal study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Wuhan University (No.2018112).

RT-qPCR

Total RNA from testes or spermatocytes was extracted using TRIzol reagent (15596026, Thermo Fisher, Waltham, MA, USA). Reverse transcription was performed using the PrimeScript RT Reagent Kit (RR037A; TaKaRa, Shiga, Japan). For real-time reverse transcription-quantitative (RT-q)PCR, primers were designed using the real-time PCR primer design service provided at https://www.genscript.com.cn/. Relative gene expression levels were assessed using the 2^-∆∆Ct method and normalized to Actb as the internal reference control. The primers used for RT-qPCR are listed in Supplementary Table 1.

In vitro fertilization and embryo culture

Sperm were collected from the epididymides of male mice and allowed to capacitate for 1 h in human tubal fluid (HTF) medium (MR-070-D; Merck Millipore, Darmstadt, Germany). Cumulus-oocyte complexes (COCs) were obtained from oviduct ampullae of female mice. Next, capacitated sperm were added to HTF drops containing COCs for fertilization in a 37°C, 5% CO₂ incubator. After co-incubation for 6 h, the COCs were washed to remove cumulus cells and excess sperm, and then transferred to KSOM (MR-101-D, Merck Millipore) for further embryo culture. The developmental potential of the early embryos was assessed at the indicated time points during culture. Relevant rates: 2PN rate = number of 2PN eggs/total number of retrieved eggs. 2-cell rate = number of 2-cell embryos / number of 2PN eggs. Blastocyst formation rate = number of blastocysts/number of 2-cell embryos.

Western blot analysis

Total protein was extracted from different tissues using RIPA buffer containing a protease inhibitor cocktail (PIC) (P6730, Solarbio, Beijing, China), followed by separation on a 10% SDS-PAGE gel. Subsequently, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH00010; Merck Millipore). The membranes were incubated with 5% nonfat milk blocking solution for 1 h at room temperature and then incubated with an anti-YBX1 antibody (1:1000 dilution, A3534, ABclonal, Wuhan, China) at 4°C overnight or with an anti-GAPDH antibody (1:6000 dilution, 60004-1-Ig, Proteintech, Wuhan, China), which was used as the loading control.

Subsequently, the membranes were incubated with goat anti-rabbit/mouse IgG-HRP secondary antibody (1:8000 dilution, SA00001-2/SA00001-1; Proteintech) for 1 h at room temperature. The protein bands were visualized using a G: BOX Chemi XRQ chemiluminescence imaging system (Syngene, Cambridge, UK) using SuperSignal TM West Pico PLUS Stable Peroxide (34577; Thermo Fisher).

Histological analysis, immunostaining, and imaging

Histological analysis, frozen section immunostaining, and spermatocyte spreading were performed as described previously [2, 9]. The primary antibodies used in this study were as follows: rabbit anti-SYCP3 (1:100, laboratory-made in the Luo laboratory, Wuhan University, China), mouse anti-γH2AX (1:100, K001451M, Solarbio), and rabbit anti-YBX1 (1:100, A3534, ABclonal). Secondary antibodies were used at a 1:200 dilution: CoraLite488- or CoraLite594-conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1, SA00013-3, Proteintech) and CoraLite488-conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2, Proteintech). An Axio Imager 2 microscope (Carl Zeiss, Oberkochen, Germany) was used to capture histological and immunostaining images.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were performed using the TMR (red) TUNEL Cell Apoptosis Detection Kit (G1502, Servicebio, Wuhan, China). The analysis was performed according to the manufacturer’s instructions. Briefly, the samples were incubated with TUNEL detection solution after permeabilization. Nuclei were labeled with 4’,6-diamidino-2-phenylindole (DAPI, 0100-20; SouthernBiotech, Birmingham, MA, USA).

Fertility assessment

To investigate the potential impact of Ybx1 deletion on fertility, adult male C57BL/6 mice (approximately 2 months old), both wild-type (WT) and Ybx1 cKO, were individually paired with an age-matched WT female (approximately 2 months old) in separate cages. These breeding pairs were maintained for at least 3 months to allow for sufficient mating opportunities. The number of pups born to each female was recorded and the average number of pups per litter was calculated by dividing the total number of pups born by the number of successful litters. This analysis aimed to determine whether Ybx1 cKO males exhibit any changes in fertility compared to their WT counterparts.

Sperm count, motility and morphology analysis

Cauda epididymides were harvested and minced in a 1.5 ml tube containing 1 ml of human tubal fluid (HTF) media and then incubated for 30 min at 37°C. Sperm count and motility were analyzed using a computer-aided sperm analysis (CASA) system. Briefly, a 20-µl aliquot of the sperm suspension was used for analysis. All sperm cells were observed using a phase-contrast microscope (Leica, Wetzlar, Germany), and videos were captured using a micromedical camera system (LABB C1220L, Beionmed, Beijing, China). The videos were processed using a sperm quality analysis system (BEION V4.20, Beionmed) to obtain different velocity parameters.

To assess sperm morphology, sperm smears were prepared, fixed, and stained using a Diff-Quick Stain Kit (G1540, Solarbio) according to the manufacturer’s protocol. The percentage of morphologically normal sperm was quantified using a bright-field optical microscope (Carl Zeiss).

RNA sequencing and analysis

RNA sequencing was performed at Benagen Company Ltd., Wuhan, China (www.benagen.com). Total RNA was extracted from isolated spermatocytes (two biological replicates for both the control and Ybx1-cKO groups). The mRNA libraries were constructed using the NEBNext® UltraTM RNA Library Prep Kit for Illumina®, followed by sequencing on the NovaSeq 6000 platform (Illumina, San Diego, CA, USA). For the gene expression analysis, the false discovery rate (FDR) was calculated and used to filter significantly different genes. Differential gene expression analysis was conducted using the R software (v.3.5.1) and the DESeq2 package. Genes were considered differentially expressed if they met the criteria of FDR < 0.05 and a fold change > 2. The datasets presented here have been submitted to online repositories. The names of the repository/repositories and their accession numbers (s) can be found at https://www.ncbi.nlm.nih.gov/ and PRJNA1079916.

Statistical analysis

All the data are expressed as the means ± standard deviations (SDs). Statistical comparisons between groups were conducted using Student’s t-test with GraphPad Prism 8.0.1. Statistical significance was set at P < 0.05.

Results

YBX1 expression during spermatogenesis in mice

To investigate the potential role of YBX1 in spermatogenesis, we first assessed the protein levels of YBX1 in multiple adult mouse tissues. YBX1 was expressed at higher levels in the testes, ovaries, spleen, and lungs (Fig. 1A). We then assessed the expression of YBX1 in developing postnatal testes and determined that the YBX1 protein was continually expressed from postnatal day 3 (P3) to adult testes (P56) (Fig. 1B). Next, we determined the localization of YBX1 in spermatogenic cells by immunofluorescence (IF) staining of testes. YBX1 was highly expressed in the cytoplasm of pachytene spermatocytes and expressed at low levels in round spermatids (Fig. 1C). High expression of YBX1 in spermatogenic cells indicates a possible role for YBX1 in mouse spermatogenesis.

Fig. 1.

High expression of YBX1 in the testis of mice. (A) Western blot analysis of YBX1 expression in different tissues from adult mice. GAPDH was used as the loading control. (B) Western blot analyses of YBX1 protein levels in mouse testes on different postnatal days. (C) Immunostaining of testis sections from adult mice with a YBX1 antibody counterstained with DAPI (blue) and γH2AX antibody indicating the stage of spermatocytes. Scale bars: 50 µm.

Conditional deletion of Ybx1 in mice

Because Ybx1-deficient embryos die during late embryonic development [19], we specifically knocked out Ybx1 in male mouse germ cells to determine the physiological roles of YBX1 in spermatogenesis. Ybx1 conditional knockout mice were generated by crossing Ybx1-floxed mice with Stra8-Cre Tg mice (Fig. 2A). Cre is expressed in the spermatogonia of Stra8-Cre mice 3 d postpartum (dpp) [20].

Fig. 2.

Generation of Ybx1 cKO mice. (A) Generation of Ybx1 cKO mice. In Ybx1 cKO mice, exons four to six were flanked by loxP for deletion by Cre/loxP-mediated recombination. (B) Western blot analysis of YBX1 protein levels in WT and Ybx1 cKO adult testes. GAPDH served as a loading control. (C) Immunostaining of testis sections from WT and Ybx1 cKO mice on postnatal day 20 using antibodies against YBX1 and γH2AX. Nuclei were stained with DAPI. Scale bars: 50 µm.

The genotype of Ybx1 cKO mice was determined through PCR genotyping of tail genomic DNA (Supplementary Fig. 1). The absence of YBX1 protein in male germ cells from Ybx1 cKO mice at P18 was confirmed using indirect IF and western blotting (Figs. 2B and C), indicating that Ybx1 cKO mice were successfully generated.

YBX1 is dispensable for meiosis but is required for spermatid development

Adult Ybx1 cKO male mice exhibited normal behavior. Although the testes were slightly smaller than the control testes, the testis-to-body weight ratio was not significantly lower than that of age-matched WT males at 2 months of age (Figs. 3A and B). Interestingly, the sperm count and motility of Ybx1 cKO male mice were significantly lower than those of WT mice (Figs. 3C and D). Other CASA-measured kinematic parameters of sperm from Ybx1 cKO mice (such as VSL, VCL, and ALH) were also reduced compared to those of sperm from WT mice (Supplementary Table 2). Diff-Quick staining revealed that the sperm from Ybx1 cKO males exhibited normal morphology (Fig. 3E). In addition, all stages of spermatogenic cells, including spermatogonial stem cells, primary spermatocytes, secondary spermatocytes, spermatids, and mature spermatozoa, were observed in testicular sections from 10-week-old WT mice and Ybx1 cKO male mice using hematoxylin and eosin (H&E) staining (Fig. 3F). However, a spermatid-shaped cluster was observed in the center of a few Ybx1 cKO seminiferous tubules, which were absent in WT tubules (Fig. 3F). Furthermore, meiotic progression was evaluated based on chromosomal spread. Chromosome spreads of spermatocytes were stained with antibodies against SYCP3 and γH2AX. SYCP3 is a component of the axial elements of the synaptonemal complex, which indicates the spermatocyte stage. γH2AX is a marker of double-stranded DNA breaks (DSBs). A similar γH2AX signal pattern was observed between WT and Ybx1 cKO spermatocytes at different stages (Supplementary Fig. 2A), indicating that DSB formation and repair were normal in Ybx1 cKO spermatocytes. Quantification of spermatocytes at different stages indicated that meiosis of Ybx1 cKO spermatocytes progressed normally (Supplementary Fig. 2B). Moreover, the number of elongated spermatids in the seminiferous tubules of 4-month-old Ybx1 cKO mice at stages IX-X was significantly lower than that in the seminiferous tubules of WT mice (Figs. 3G and H), and there were no sperm in some of the tubules (marked with asterisks) observed through H&E staining of the epididymis of 4-month-old Ybx1 cKO mice (Fig. 3I), indicating that YBX1 plays an essential role in spermatid development. Finally, a TUNEL assay was used to determine the influence of Ybx1 cKO on germ cell apoptosis. The number of apoptotic cells per tubule was comparable between WT and Ybx1 cKO testes (Supplementary Fig. 3), showing that the deletion of Ybx1 did not result in germ cell apoptosis. Collectively, these findings indicate that Ybx1 is dispensable for meiosis, but is required for spermatid development.

Fig. 3.

Ybx1 deletion leads to impaired spermiogenesis. (A) Representative images of testes from 10-week-old WT and Ybx1 cKO mice. (B) Testis to body weight ratios in 10-week-old WT and Ybx1 cKO mice (n = 3). (C) Sperm counts of 4-month-old WT and Ybx1 cKO mice (n = 3). (D) Total sperm motility of WT and Ybx1 cKO mice (n = 3). (E) Sperm morphology and percentage of normal sperm morphology in WT and Ybx1 cKO mice. (F) H&E staining of testes from 10-week-old WT and Ybx1 cKO mice. The black arrow indicates a spermatid-shaped cluster. Scale bars: 100 µm. (G) IF of γH2AX (green), PNA (red) and DAPI (blue) of 4-month-old seminiferous tubules at stage IX-X from WT and Ybx1 cKO mice. Scale bars: 50 µm. (H) Elongated spermatid quantification in the seminiferous tubules of 4-month-old WT and Ybx1 cKO mice at stage IX-X. (I) H&E staining of epididymis from 4-month-old WT and Ybx1 cKO mice. ns, no significant difference. *** P < 0.0001. Scale bars: 200 µm.

Ybx1 deficiency decreases the fertilization potential of sperm in vitro

To investigate whether Ybx1 deficiency affected male fertility, we performed mating tests and showed that Ybx1 cKO males were fertile. The results of the mating tests indicated that Ybx1 cKO males were hypofertile, with a significant reduction in the average number of pups compared to WT males (Fig. 4A), indicating that Ybx1 deletion decreased the fertility of male mice.

Fig. 4.

Ybx1 deletion impaired the fertilization potential of spermatozoa. (A) Average number of pups per litter from the WT and Ybx1 cKO groups (mean ± SD). (B) MII oocytes were fertilized with spermatozoa from adult WT and Ybx1 cKO male mice. The zygotes, which contained two pronuclei, were observed at 6 h after fertilization. The two-cell embryos were observed at approximately 24 h after fertilization. The blastocysts were observed at approximately 94 h after fertilization. Scale bars: 100 µm. (C) The percentages of 2PN zygotes, two-cell stage embryos, and blastocysts after IVF with WT or Ybx1 cKO sperm was calculated. ns, no significant difference, * P < 0.01, *** P < 0.0001.

To determine the fertilization potential of Ybx1 cKO spermatozoa, we conducted an in vitro fertilization (IVF) assay. MII oocytes were collected from the ampullary region of the oviducts and used for IVF. The fertilization efficiency was measured by observing two pronuclei, two-cell embryos, and blastocysts. We observed a significantly lower proportion of 2 pronucleus zygotes following IVF with Ybx1 cKO sperm than in IVF with WT spermatozoa (Figs. 4B and C), indicating that Ybx1 deficiency decreased the fertilization potential of spermatozoa in vitro.

Discussion

In this study, we determined the role of YBX1 in male fertility and spermatogenesis using Ybx1 cKO mouse model. Our results demonstrate that YBX1 plays a vital role in sperm quality and reproductive capacity, as indicated by the parameters of sperm from the epididymis and the decline in fertility. Interestingly, although YBX1 was highly expressed in the testes and predominantly in the cytoplasm of pachytene spermatocytes, meiosis was normal in Ybx1 cKO male mice. Notably, the number of elongated spermatids was significantly reduced in Ybx1 cKO tubules.

A previous study revealed that the loss of YBX2 leads to the degeneration of spermatids and vacuolation of germ cell cytoplasm, causing male infertility [21]. YBX3 reduces male fertility by increasing spermatocyte apoptosis and seminiferous tubule degeneration [22]. In this study, Ybx1 deficiency reduced male fertility by impairing spermatid development and sperm function but did not increase spermatocyte apoptosis. Therefore, although YBX1, YBX2, and YBX3 are members of the cold-shock domain family, the importance of YBX1 and YBX3 in male fertility is lower than that of YBX2. Mechanistically, YBX1 is expressed at a relatively lower in round spermatid compared with YBX2 expression, which promotes mRNA storage by repressing mRNA translation and blocking translation-dependent mRNA decay [23]. Therefore, we speculated that the function of YBX1 could be partially compensated for by YBX2.

In our study, although meiosis progressed normally in Ybx1 cKO spermatocytes without significant differences in apoptosis, the number of sperm from Ybx1 cKO epididymis was significantly reduced. To interpret the phenotype, different stages of germ cells were calculated and compared between the WT and Ybx1 cKO tubules. Our results showed no significant differences in the numbers of leptotene/zygotene and pachytene spermatocytes between WT and conditional knockout (cKO) seminiferous tubules. However, the number of round and elongated spermatids in cKO seminiferous tubules was significantly lower than that in their WT counterparts, while the number of primary spermatocytes remains normal in seminiferous tubules (Figs. 3G, 3H and Supplementary Fig. 4). Therefore, our results demonstrate that Ybx1 deficiency results in defects in the post-meiotic stage.

According to previous studies, YBX1 is a multifunctional DNA/RNA binding protein that participates in regulating pre-mRNA splicing, mRNA transcription, stability, and translation [16, 24, 25]. Because YBX1 is highly expressed in spermatocytes, RNA-seq was performed using WT and Ybx1 cKO spermatocytes. Our results showed that Ybx1 deficiency had a relatively minor effect on RNA transcription and splicing (Supplementary Fig. 5). Considering the role of YBX1 in regulating mRNA translation in oocytes [16, 26], we speculated that sperm defects resulted from abnormal mRNA translation in Ybx1 cKO germ cells.

A recent study reported that Ybx1 knockdown did not significantly change the expression of YBX2 or YBX3 at the 8-cell stage [3]. Here, Ybx1 deletion had no effect on the transcription of Ybx2 or Ybx3 during spermatogenesis (Supplementary Fig. 6). Therefore, YBX1 does not regulate the expression of other YBX members during spermatogenesis or the development of re-implantation embryos. Although YBX1 interacts with MEIOB and PPRC2A, two proteins critical for meiosis I completion [5], Ybx1 deletion has no impact on meiosis progression. Therefore, we concluded that the interaction of YBX1 with MEIOB and PPRC2A is not essential for their roles of MEIOB and PPRC2A.

In summary, we analyzed the phenotype of Ybx1 cKO male mice and demonstrated that YBX1 is dispensable for meiosis, although YBX1 is highly expressed in the cytoplasm of pachytene spermatocytes. Ybx1 deletion leads to subfertility in males, which can result from impaired spermatid development and decreased fertilization potential in Ybx1 cKO sperm. Therefore, our results provide basic information for related studies and help to elucidate the role of YBX1 in male fertility.

Conflict of interests

The authors declare that this study was conducted in the absence of commercial or financial relationships that could be construed as potential conflicts of interest.