The Loss-Function of KNL1 Causes Oligospermia and Asthenospermia in Mice by Affecting the Assembly and Separation of the Spindle through Flow Cytometry and Immunofluorescence

KNL1 (kinetochore scaffold 1) has attracted much attention as one of the assembly elements of the outer kinetochore, and the functions of its different domains have been gradually revealed, most of which are associated with cancers, but few links have been made between KNL1 and male fertility. Here, we first linked KNL1 to male reproductive health and the loss-function of KNL1 resulted in oligospermia and asthenospermia in mice (an 86.5% decrease in total sperm number and an 82.4% increase in static sperm number, respectively) through CASA (computer-aided sperm analysis). Moreover, we introduced an ingenious method to pinpoint the abnormal stage in the spermatogenic cycle using flow cytometry combined with immunofluorescence. Results showed that 49.5% haploid sperm was reduced and 53.2% diploid sperm was increased after the function of KNL1 was lost. Spermatocytes arrest was identified at the meiotic prophase I of spermatogenesis, which was induced by the abnormal assembly and separation of the spindle. In conclusion, we established an association between KNL1 and male fertility, providing a guide for future genetic counseling regarding oligospermia and asthenospermia, and a powerful method for further exploring spermatogenic dysfunction by utilizing flow cytometry and immunofluorescence.


Introduction
For eukaryotic cells, the process of accurate chromosome separation is described as a delicate instrument that each slight error may cause dysregulation of cell proliferation or even disease phenotype [1][2][3][4]. The kinetochore is a macromolecular protein complex that assembles at centromeres and connects to spindle microtubules [5][6][7]. It also performs several indispensable functions in the cell-division process by linking the centromere and microtubule, providing a platform for the spindle assembly checkpoint (SAC) protein [8][9][10][11]. More than 100 kinetochore proteins from yeast to mammalian cells have been identified [8]. The study of kinetochore compositions is significant and necessary to shed light on this intriguing complex in multiple fields concerning functions, evolutions, and diseases.
KNL1 (also known as CASC5, D40, Blinkin) [12] is one important member required for genomic stability in eukaryotes in the conserved KMN network (KNL1 complex, NDC80 complex, Mis12 complex), constituting the core microtubule-binding site at the kinetochore [13][14][15]. KNL1 regulates cells by coordinating multiple protein-protein interactions Table 1. Part of the literature on the changes of FCM used in sperm detection.

Researchers
Year Methods

CYAN-ADP Sperm capacitation and functions
Evelyn et al. [36] 2023 FCM + co-staining SytoxGreen™ and dihydroethidium (DHE) Amnis ® ImageStream ® Certain morphologic abnormalities and ROS Here, we first found that the loss of KNL1 caused oligospermia and asthenia in mice and detected the abnormal composition of different ploidy sperm cells by FCM. The knockout of KNL1 could lead to a decrease in the immunofluorescence signal of GFRα1, PLZF, γH2AX, and Sycp3 by employing IF staining, which means the spermatocytes arrested at the meiotic prophase I of spermatogenesis. Moreover, the spindle was unable to maintain its polarity and alignment correctly in spermatocytes in GC-2 cells, which resulted in the assembly separation of the spindle and confirmed that the method of FCM plus IF was useful in detecting the periodic location of spermatogenesis problems. In a word, our study not only provides a powerful method for spermatogenic dysfunction by FCM and IF but also exposes the relationship between KNL1 and reproductive development, which helps to determine genotypic-phenotypic associations as well as improve the diagnosis rate of male infertility.

Cell Lines and Cell Culture
GC-2 (CEMC, Sydney NSW, Australia) cells were purchased from the Center for Excellence in Molecular Cells Science and cultured in DMEM (ThermoFisher, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (Gibco, Grand Island, Australia) and 1% Penicillin-Streptomycin Solution (Gibco, Grand Island, Australia). All cells used in experiments were cultured at 37 • C in a 5% CO 2 incubator.

Mice Culture
Sexually mature male ICR mice (more than four weeks old) were purchased from Charles River Laboratory Animal Technology CO., Ltd. (Wilmington, MA, USA) The mice were placed in a specific pathogen-free animal room under controlled conditions (temperature, 25 ± 2 • C, humidity (50-60%), and a 12/12 h light/dark cycle. All mice were acclimated to the culture environment for at least one week before formal experiments. After the completion of the operation, the mice were executed after two spermatogenic processes, and the subsequent experiments continued on the mice.

Seminiferous Tubule Injection Experiment
ICR Mice were anesthetized by intraperitoneal injection (sodium pentobarbital). On the microoperation table, the reproductive organs were exposed with surgical scissors and surgical tweeds, and KNL1 siRNA (60 µg) mixed with Entranster in vivo (Engreen Biosystem, Beijing, China) solution, added with bromophenol blue as an indicator, was injected into the seminiferous tubules of the mice. After completion of the operation, the wounds of the mice were sutured.

Sample Processing for HE Staining and CASA
After two spermatogenic cycles, mice were executed and testes were removed for the first recording of phenotypic data such as length, width, and surface area. The testis and epididymis were collected and placed in a fixative solution, sectioned, and stained with HE.
The epididymis was collected and cut, and the sperm was extruded by gently squeezing the epididymis with ophthalmic forceps. Then, the mixture was incubated at 37 • C, 5% CO 2 , and saturated humidity for 20 min. After the semen was completely liquefied, the mixture was evenly added to the corresponding counting board for detection for CASA detection (Hamilton Thorne, Beverly, MA, USA, TVOS) using the following parameters: VCL cutoff of 36 M/s, VSL cutoff of 5 µm/s, VAP cutoff of 20 µm, progressive minimum PR cutoff of 32%. At least 200 sperm were analyzed, and the results of the two analyses passed the statistical 95% CI. The remaining testes and epididymis tissues, which were left over after the experiment above, were frozen at −20 • C.

Flow Cytometry Experiment
The testis was dissected out, precooled, and washed once with 1X PBS (Solarbio, Beijing, China). We removed the tunica albuginea of the testis and fully cut up testicular tissue into the EP tube. Type II collagenase (Life-iLab, Shanghai, China) was added in 1 mL DMEM/F12 to a final concentration of 1.5 mg/mL, and the chopped testicular tissue was transferred to 37 • C and digested with 50 r/min shaking for 20 min. Then, the supernatant was removed by centrifugation at 1500 r/min for 5 min. Type II collagenase and hyaluronidase (MERK, Kenilworth, NJ, USA) were added to 0.5% trypsin to a final concentration of 1.5 mg/mL. We added 500 µL to resuspend cell mass and digested for 20-30 min at 37 • C with shaking at 150 r/min. Digestion was terminated by adding 500 µL DMEM/F12 and centrifuging at 1500 r/min for 5 min to remove the supernatant. We added 500 µL PBS (containing RNaseA (50 µg/mL)) and incubated at 37 • C for 30 min, and centrifuged at 1500 r/min for 5 min to remove the supernatant. The samples were washed once with PBS and centrifuged at 1500 r/min for 5 min to remove the supernatant.
The working mixture of Tritonx-100 + PI (biosharp, Shanghai, China) was added: PI to the final concentration of 50 µg/mL, Tritonx-100 (Sigma-Aldrich, Saint Louis, MO, USA) to the final concentration of 0.03%, and cells were drilled and stained for 10 min at room temperature in the dark. After all the treatment, the mixture was filtered with a 200 mesh filter and test on the machine.

TUNEL and PNA Method
The mice were killed by cervical dislocation, and the needed testicular tissue was removed and cut into a small piece 2-3 mm thick to facilitate the penetration of the fixative. The cut testicular tissue was washed with 1X PBS, fixed in the testicular tissue fixation fluid (Servicebio, Wuhan, China), and sent to the Servicebio Company (Wuhan, China) for TUNEL and PNA detection.

Immunofluorescence Staining
The sections or cell slides were washed with 1X PBS. They were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 30 min at room temperature and washed three times with 1X PBS for 3 min each time. Membranes were permeabilized with Triton X-100 for 30 min at room temperature and washed three times with PBS for 3 min each. The cells were blocked overnight at 4 • C in 3% BSA (Solarbio, Beijing, China). The primary antibody was diluted, incubated at 37 • C for 1 h, and washed three times with 1X PBS for 5 min each time. The secondary antibody was diluted, incubated at 37 • C for 1 h, and washed three times with PBS for 5 min each time. Nuclear staining was performed with DAPI (Beyotime, Shanghai, China) and observed with an Olympus FV 3000.

siRNA Transfection and Synchronization
The cells were seeded into six-well plates a day earlier to maintain a cell density of 60%-80%. Then, 125 µL DMEM medium without antibiotics and serum was added to the EP tubes, followed by 100 pmol siRNA, blowing and mixing, then 4 µL Lipo8000™ (Beyotime, Shanghai, China) transfection reagent was added, blowing and mixing to mix well, and placed at room temperature for 20 min. Drops were added to six-well plates for cellular siRNA interference. Some cells were used for qRT-PCR to detect the efficiency of KNL1 interference. For synchronization, after 16 h of interference in the above siRNA interference, the cells were cultured with 10 mM thymidine (Tsbiochem, Shanghai, China) for 16 h. Then, we released the thymidine to replace the medium as new and the immunofluorescence observation was performed 10 h later.

siRNA Sequence and Immunofluorescence Antibody
The siRNA sequence used in the cell interference experiment is Mus-siKNL1(5 -3 : UCGAGUCAGCUUUGCAGAUACUAUA) ordered from GENEray Biotechnology. The qRT-PCR sequence used in the experiment is KNL1-F: CAAAACCGAAAACTGCAGGGC; KNL1-R: TTTGGCTCAAGACAGCTTACC. The immunofluorescence antibody in the experiment was shown in the Supplement data Table S1.

The Loss-Function of the KNL1 Gene Influences Male Reproductive Phenotype in Mice
We extracted the expression of the KNL1 gene from the single-cell RNA-seq data database provided by Tong's team, which uncovered dynamic processes in mouse spermatogenesis, revealing extensive and previously uncharacterized dynamic processes and molecular markers in gene expression [37]. Interestingly, we found that the gene KNL1

The Loss-Function of the KNL1 Gene Influences Male Reproductive Phenotype in Mic
We extracted the expression of the KNL1 gene from the single-cell RNA-seq d tabase provided by Tong's team, which uncovered dynamic processes in mouse sp ogenesis, revealing extensive and previously uncharacterized dynamic processes an lecular markers in gene expression [37]. Interestingly, we found that the gene KN pression is extremely high during spermatogenesis (Figure 1), implicating that the gene might play an important role in spermatogenesis.

Figure 1.
Log2 TPM values in different spermatogenic stages. A1 represents the type A1 sp gonia. In represents the Intermediate spermatogonia. BS represents the Type B spermato phase. BG2 represents the Type B spermatogonia G2M phase. G1 represents the G1 phas preleptotene spermatocyte stage. ePL represents the early phase of the preleptotene sperm stage. mPL represents the medium-term phase of the preleptotene spermatocyte stage. IPL sents the late phase of the preleptotene spermatocyte stage. L represents the leptotene stage osis. Z represents the even line stage of meiosis. eP represents the early pachytene of meio represents the metaphase pachytene of meiosis. IP represents the late pachytene of meiosis resents the double-line stage of meiosis. MI represents the first meiotic division. MII repres second meiotic division. RS1-2 represents the round spermatid stage 1-2. RS3-4 represe round spermatid stage 3-4. RS5-6 represents the round spermatid stage 5-6. RS7-8 represe round spermatid stage 7-8.
We exposed the testis to injected siRNA (60 µg) in seminiferous tubules of IC to influence the KNL1 expression in the testis (Figure 2A). After two whole spermat cycles, we executed mice that received no interference (KNL1-Ctrl mice) and KNL1 iment mice (KNL1-60 µg mice) to test different phenotype indicators. We found KNL1-60 µg mice, the size of the testis tended to decrease a little compared to KN mice ( Figure 2B,C).

Figure 1.
Log2 TPM values in different spermatogenic stages. A1 represents the type A1 spermatogonia. In represents the Intermediate spermatogonia. BS represents the Type B spermatogonia S phase. BG2 represents the Type B spermatogonia G2M phase. G1 represents the G1 phase of the preleptotene spermatocyte stage. ePL represents the early phase of the preleptotene spermatocyte stage. mPL represents the medium-term phase of the preleptotene spermatocyte stage. IPL represents the late phase of the preleptotene spermatocyte stage. L represents the leptotene stage of meiosis. Z represents the even line stage of meiosis. eP represents the early pachytene of meiosis. mP represents the metaphase pachytene of meiosis. IP represents the late pachytene of meiosis. D represents the double-line stage of meiosis. MI represents the first meiotic division. MII represents the second meiotic division. RS1-2 represents the round spermatid stage 1-2. RS3-4 represents the round spermatid stage 3-4. RS5-6 represents the round spermatid stage 5-6. RS7-8 represents the round spermatid stage 7-8.
We exposed the testis to injected siRNA (60 µg) in seminiferous tubules of ICR mice to influence the KNL1 expression in the testis ( Figure 2A). After two whole spermatogenic cycles, we executed mice that received no interference (KNL1-Ctrl mice) and KNL1 experiment mice (KNL1-60 µg mice) to test different phenotype indicators. We found that in KNL1-60 µg mice, the size of the testis tended to decrease a little compared to KNL1-Ctrl mice ( Figure 2B,C).
The HE staining section of the testis, testicular biopsy, and sperm between KNL1-Ctrl mice and KNL1-60 µg mice revealed changes in phenotypic characteristics. The testicular biopsy showed the defect of germ cell development in KNL1-60 µg mice compared to KNL1-Ctrl mice ( Figure 3A). It was obvious that KNL1-Ctrl mice testis sections showed normal cell populations within the seminiferous epithelium, whereas germ cells lost most of the important developmentally relevant cells in KNL1-60 µg mice testis. Similarly, in the epididymal, HE staining section ( Figure 3B), a marked decrease in sperm count was observed in KNL1-60 µg mice. Dramatically, the KNL1-60 µg mice had no mature spermatozoa or round spermatozoa compared with KNL1-Ctrl mice ( Figure 3C). The HE staining section of the testis, testicular biopsy, and sperm between KNL1-Ctrl mice and KNL1-60 µg mice revealed changes in phenotypic characteristics. The testicular biopsy showed the defect of germ cell development in KNL1-60 µg mice compared to KNL1-Ctrl mice ( Figure 3A). It was obvious that KNL1-Ctrl mice testis sections showed normal cell populations within the seminiferous epithelium, whereas germ cells lost most of the important developmentally relevant cells in KNL1-60 µg mice testis. Similarly, in the epididymal, HE staining section ( Figure 3B), a marked decrease in sperm count was observed in KNL1-60 µg mice. Dramatically, the KNL1-60 µg mice had no mature spermatozoa or round spermatozoa compared with KNL1-Ctrl mice ( Figure 3C).   The HE staining section of the testis, testicular biopsy, and sperm between KNL1-Ctrl mice and KNL1-60 µg mice revealed changes in phenotypic characteristics. The testicular biopsy showed the defect of germ cell development in KNL1-60 µg mice compared to KNL1-Ctrl mice ( Figure 3A). It was obvious that KNL1-Ctrl mice testis sections showed normal cell populations within the seminiferous epithelium, whereas germ cells lost most of the important developmentally relevant cells in KNL1-60 µg mice testis. Similarly, in the epididymal, HE staining section ( Figure 3B), a marked decrease in sperm count was observed in KNL1-60 µg mice. Dramatically, the KNL1-60 µg mice had no mature spermatozoa or round spermatozoa compared with KNL1-Ctrl mice ( Figure 3C).

The Loss-Function of the KNL1 Gene Results in Oligospermia and Asthenospermia in Mice
For further research, we sought to test sperm motility by CASA (computer-aided sperm analysis) detection. The mice CASA result showed the sperm count had a significant decrease in the total count, progressiveness, and motility in the KNL1-60 µg mice (Table 2 and Figure 4A). Although there was no significant difference in the absolute number of static sperm between the two groups (Table 3, Figure 4A,B), there was also a significant increase in percentage (relative to total sperm count), which was harmful to their breeding in the future.

Loss-Function of KNL1 Causes Spermatocytes Arrest at the Meiotic Prophase I of Spermatogenesis
The above results showed a significant decrease in sperm count and quality. We examined the integrity of sperm acrosomes by detecting peanut lectin (PNA, Figure 5A,B) and the results showed that the integrity of KNL1-60 µg mice sperm acrosome was significantly reduced. Next, we performed the TUNEL experiment on the section of spermatogenic tubules to prove that the degenerative phenotype of reproduction in male mice was related to apoptosis. However, to our surprise, there was no significant difference between the KNL1-Ctrl mice and the KNL1-60 µg mice ( Figure 5C,D), which confirmed that apoptosis was not the main factor inducing reproductive degeneration phenotype of oligospermia and azoospermia, as many previously reported [38][39][40].
icantly reduced. Next, we performed the TUNEL experiment on the section of spermatogenic tubules to prove that the degenerative phenotype of reproduction in male mice was related to apoptosis. However, to our surprise, there was no significant difference between the KNL1-Ctrl mice and the KNL1-60 µg mice ( Figure 5C,D), which confirmed that apoptosis was not the main factor inducing reproductive degeneration phenotype of oligospermia and azoospermia, as many previously reported [38][39][40].  Considering the FC results and the spermatogenic process, we speculated that KNL1 influenced the process of meiosis of spermatogenesis, leading to the accumulation of diploid (2N) and tetraploid (4N) sperm cells and the failure of haploid (N) sperm production.
To precisely confirm which stage the KNL1 would affect in the spermatogenic cycle, we chose different fluorescence markers to map the cycle of KNL1 influenced during spermatogenesis. By the detection of fluorescence intensity of different markers such as GFRα1, PLZF, C-kit, STRA8, γH2AX, and Sycp3, we found that GFRα1, PLZF, γH2AX, and Sycp3 had significant differences in mouse testis between the KNL1-60 µg group and KNL1-Ctrl mice ( Figures 7A-H and S2). Considering the FC results and the spermatogenic process, we speculated influenced the process of meiosis of spermatogenesis, leading to the accumulat loid (2N) and tetraploid (4N) sperm cells and the failure of haploid (N) sperm p To precisely confirm which stage the KNL1 would affect in the spermatog we chose different fluorescence markers to map the cycle of KNL1 influenced d matogenesis. By the detection of fluorescence intensity of different marke GFRα1, PLZF, C-kit, STRA8, γH2AX, and Sycp3, we found that GFRα1, PLZF, γ Sycp3 had significant differences in mouse testis between the KNL1-60 µg KNL1-Ctrl mice ( Figures 7A-H and S2). These results again confirmed that the damage caused by KNL1 deletion to spermatogonia mainly lies in the meiosis stage, especially affecting the transition stage of the meiosis I stage and partially affecting mitosis. We thus put forward the hypothesis that the oligospermia of KNL1 mice mainly resulted from the loss-function of KNL1 on spermatocyte meiosis, resulting in the block of the maturation process of spermatocytes.

The Loss-Function of KNL1 Leads to Abnormal Assembly and Separation of the Spindle Resulting in Unequal Cell Segregation
To further investigate the effect of KNL1 on spermatocyte maturation, GC-2 cells that could maintain the stage of spermatocyte were selected for this study. The expression level of KNL1 in GC-2 cells was knocked down by siRNA ( Figure S3A), and the GC-2 cells were treated with paving and immunofluorescence after interference. The results showed that GC-2 cells treated with KNL1 siRNA exhibited unequal division compared to normal GC-2 cells (Figure 8). These results again confirmed that the damage caused by KNL1 deletion to spermatogonia mainly lies in the meiosis stage, especially affecting the transition stage of the Testis was immunostained with anti-γH2AX (green). Merge was DAPI (blue) and γH2AX (green). (D) SYCP3 fluorescent images of testis in KNL1-Ctrl and KNL1-60 µg. Testis was immunostained with anti-SYCP3 (red). Merge was DAPI (blue) and SYCP3 (red). (E-H) Data were presented as mean percentages (mean ± SEM) of at least three independent measurements. Asterisk denotes statistical difference at a p (**) < 0.0 1, p (****) < 0.0001 level of significance. Bar = 50 µm.
To further investigate the effect of KNL1 on spermatocyte maturation, GC-2 cells that could maintain the stage of spermatocyte were selected for this study. The expression level of KNL1 in GC-2 cells was knocked down by siRNA ( Figure S3A), and the GC-2 cells were treated with paving and immunofluorescence after interference. The results showed that GC-2 cells treated with KNL1 siRNA exhibited unequal division compared to normal GC-2 cells (Figure 8). Considering the previous reports of the KNL1 gene in the literature, we first speculated that KNL1 may lead to unequal division by affecting the spindle. We also transfected cells with siRNA to interfere with the expression of KNL1. Immunofluorescence results showed that compared with the non-interference group, the spindle in the KNL1-60 µg group represented different degrees of abnormality compared to the KNL1-Ctrl group ( Figure S3), such as the emergence of multipolar spindles in the metaphase and the failure of spindle formation ( Figures 9A,B and S3B,C).
In addition, we also observed the phenomenon of chromosomal abnormalities in interfered cells. Chromosomes were not fully aligned on the equatorial plate in metaphase, and there was a phenomenon of chromosome lag in anaphase ( Figure 9C,D). Considering the previous reports of the KNL1 gene in the literature, we first speculated that KNL1 may lead to unequal division by affecting the spindle. We also transfected cells with siRNA to interfere with the expression of KNL1. Immunofluorescence results showed that compared with the non-interference group, the spindle in the KNL1-60 µg group represented different degrees of abnormality compared to the KNL1-Ctrl group ( Figure S3), such as the emergence of multipolar spindles in the metaphase and the failure of spindle formation ( Figures 9A,B and S3B,C).
In addition, we also observed the phenomenon of chromosomal abnormalities in interfered cells. Chromosomes were not fully aligned on the equatorial plate in metaphase, and there was a phenomenon of chromosome lag in anaphase ( Figure 9C,D). We inferred that the disorder of the spindle leads to the decline of its ability to regulate chromosomes and the spindle cannot regulate chromosomes as accurately as before, which resulted in the abnormal arrangement of some chromosomes based on the evidence We inferred that the disorder of the spindle leads to the decline of its ability to regulate chromosomes and the spindle cannot regulate chromosomes as accurately as before, which resulted in the abnormal arrangement of some chromosomes based on the evidence above. The force imbalance causes chromosome lag in the subsequent cell separation and leads to abnormal cell division.

Discussion
Maintaining reproductive health is crucial to successful fertility and a healthy relationship between partners, while most cases of infertility originate from the poor production of sperm or eggs [41]. Nowadays, male infertility is a big problem plaguing mankind, which may be caused by genetic factors or an unhealthy lifestyle [42]. There are several factors such as genetic mutations, infections, anatomical change, and pressure involved in the normal development and quality of sperm that stop men from being able to father offspring [43]. The phenotype of nearly 90% of male infertility is mainly reflected in sperm, including sperm quantity, quality, and activity [44].
KNL1 is an essential large scaffold protein for accurate chromosome segregation in eukaryotic cells [16]. It was first recognized as a kinetochore protein in 2004 in human cells [45]. Subsequently, more and more studies have gradually revealed the functions and corresponding connections of different domains and motifs of KNL1 [9,12,[46][47][48]. Previous studies have shown that KNL1 plays important roles in several types of cancer, and KNL1 was found to be abnormally expressed in 16 types of cancer [49,50]. However, its relationship with the male reproduction system remains largely unclear.
In our study, the expression of KNL1 in the testis was knocked down by injecting the corresponding siRNA into the seminiferous tubules of the mice to explore the influence on the male reproductive phenotype. To our surprise, no obvious changes in testicular size were observed in NC and KNL1 groups. However, when we detected the internal physiological structure of the testis and epididymis, we found the KNL1 group lost most of the important developmentally relevant cells and there is a significant decrease in the number of sperm (Tables 2 and 3 and Figure 4A-D). Compared to the control group, the quality and quantity of sperm both have a huge reduction. These phenotypes are consistent with those previous studies concerning oligospermia and asthenospermia due to gene variants, oxidative stress, or stimulation of sexual hormone secretion [51][52][53][54]. In the flow cytometry, we found that the number of diploid cells in the experimental group was significantly accumulated, suggesting that there was a block in the process of spermatogenesis, leading the number of mature sperm to decrease. At this point, these phenotypes were already consistent with the diagnosis of oligospermia and asthenospermia.
Next, the effect and underlying mechanism of this block in spermatogenesis were investigated. Previous researchers found that apoptosis might be one of the important reasons leading to sperm problems [38][39][40]. However, it was surprising to find that apoptosis was not the main factor causing sperm reduction and sperm arrest. Immunofluorescence experiments were performed to detect the markers in each stage of spermatogenesis to precisely locate the problem period and we found that the attenuation of the fluorescence signal was most obvious in the meiotic phase, which combined with the previous flow cytometry results. We believed that the problem of sperm arises in the first meiotic division. GC-2 cell as a cell model was adopted to explore the specific effect and underlying mechanism after KNL1 interference. We found that unequal division of GC-2 cells occurred after the knockdown of KNL1 expression in GC-2 cells. Since previous reports have confirmed the role of KNL1 in the regulation of kinetosomes during cell division, we hypothesized that KNL1 also has a certain effect on the spindle. Consistent with our hypothesis, there was a phenomenon of multipolar spindle and spindle dispersion after observing KNL1 knockdown cells by immunofluorescence. Additionally, chromosome disarrangement and chromosome hysteresis were also observed. Taken together, the knockdown of KNL1 could lead to decreased adhesion to the spindle, resulting in the abnormal spindle and decreased tensile force on the chromosome, abnormal division in cells, and increased probability of aneuploidy in cells. For male reproduction, it can lead to abnormal spermatogenesis, oligospermia, and asthenospermia.
To conclude, we first revealed the association between the KNL1 gene and spermatogenic dysfunction and further explored the mechanism of KNL1 in causing the occurrence of spermatogenic dysfunction. KNL1 can become a potential molecular prognostic and diagnostic marker in some cases while normal phenotypes are easily ignored. What is more, our work not only further reveals the role of the KNL1 gene, but also provides a more accurate tool for detecting the abnormal stages of spermatogenesis by using FCM and IF, as well as expanding the diagnostic pointer pool in the field of male reproduction. As a result, it can improve diagnostic accuracy if coupled with other clinical diagnostic technologies. However, some limitations should be noted. Firstly, although we identified the problem stage in the meiosis I stage, we did not pinpoint the different stages in meiosis such as the leptotene stage, or even the zygotene stage, pachytene stage, diplotene stage, and diakinesis phase. Other innovative technology to further explore more accurate positioning is worth further exploration in our experiment that we should continue to explore. Secondly, establishing an animal model of the cKO mice to explore the mechanism of KNL1 in more detail in subsequent studies should be considered. In addition, to date there has been no reported human case associated with KNL1, and future studies in the reproductive field should be paid attention to establish a strong link between KNL1 and human sperm quality and health.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to some experimental research still in progress.