Genotyping of rams based on melatonin receptor 1A gene polymorphisms: a tool in sire selection?

Context Several polymorphisms in the melatonin receptor 1A gene (MTNR1A ) have been related to reproductive performance in ovine. Aims To investigate the effect of the Rsa I and Mnl I polymorphisms on ram seminal quality. Methods Eighteen Rasa Aragonesa rams were genotyped for the Rsa I (C/C, C/T, T/T) and Mnl I (G/G, G/A, A/A) allelic variants of the MTNR1A gene. Individual ejaculates were analysed once a month throughout the whole year. Sperm motility, morphology, membrane integrity, levels of reactive oxygen species (ROS), phosphatidylserine (PS) inversion, DNA fragmentation and capacitation status were assessed. The effect of the season and polymorphisms on seminal quality was evaluated by mixed ANOVA. Key results Both polymorphisms had an effect on membrane integrity and viable spermatozoa with low levels of ROS and without PS translocation, and Rsa I also on motile and DNA-intact spermatozoa. An interaction between both polymorphisms was found, pointing to a negative effect on seminal quality of carrying the T or A allele in homozygosity. Differences were higher in the reproductive than in the non-reproductive season. Conclusions Mutations substituting C by T and G by A at Rsa I and Mnl I polymorphic sites, respectively, in the MTNR1A gene in rams could decrease the seminal quality. Implications Genotyping of rams based on melatonin receptor 1A could be a powerful tool in sire selection.

The lack of Tex44 causes severe subfertility with flagellar abnormalities in male mice.

By analyzing a mouse Interspecific Recombinant Congenic Strain (IRCS), we previously identified a quantitative trait locus (QTL), called Mafq1 on mouse chromosome 1, that is associated with male hypofertility and ultrastructural sperm abnormalities. Within this locus, we identified a new candidate gene that could be implicated in a reproductive phenotype: Tex44 (Testis-expressed protein 44). We thus performed a CRISPR/Cas9-mediated complete deletion of this gene in mice in order to study its function. Tex44-KO males were severely hypofertile in vivo and in vitro due to a drastic reduction of sperm motility which itself resulted from important morphological sperm abnormalities. Namely, Tex44-KO sperm showed a disorganized junction between the midpiece and the principal piece of the flagellum, leading to a 180° flagellar bending in this region. In addition, the loss of some axonemal microtubule doublets and outer dense fibers in the flagellum's principal piece has been observed. Our results suggest that, in mice, TEX44 is implicated in the correct set-up of the sperm flagellum during spermiogenesis and its absence leads to flagellar abnormalities and consequently to severe male hypofertility.

Genetic Causes of Qualitative Sperm Defects: A Narrative Review of Clinical Evidence.

Several genes are implicated in spermatogenesis and fertility regulation, and these genes are presently being analysed in clinical practice due to their involvement in male factor infertility (MFI). However, there are still few genetic analyses that are currently recommended for use in clinical practice. In this manuscript, we reviewed the genetic causes of qualitative sperm defects. We distinguished between alterations causing reduced sperm motility (asthenozoospermia) and alterations causing changes in the typical morphology of sperm (teratozoospermia). In detail, the genetic causes of reduced sperm motility may be found in the alteration of genes associated with sperm mitochondrial DNA, mitochondrial proteins, ion transport and channels, and flagellar proteins. On the other hand, the genetic causes of changes in typical sperm morphology are related to conditions with a strong genetic basis, such as macrozoospermia, globozoospermia, and acephalic spermatozoa syndrome. We tried to distinguish alterations approved for routine clinical application from those still unsupported by adequate clinical studies. The most important aspect of the study was related to the correct identification of subjects to be tested and the correct application of genetic tests based on clear clinical data. The correct application of available genetic tests in a scenario where reduced sperm motility and changes in sperm morphology have been observed enables the delivery of a defined diagnosis and plays an important role in clinical decision-making. Finally, clarifying the genetic causes of MFI might, in future, contribute to reducing the proportion of so-called idiopathic MFI, which might indeed be defined as a subtype of MFI whose cause has not yet been revealed.

Yoga and its effect on sperm genomic integrity, gene expression, telomere length and perceived quality of life in early pregnancy loss.

Achieving successful pregnancy outcomes is a delicate interplay between the maternal and the fetal counterparts. Paternal factors play a critical role in health and disease of offspring. Early pregnancy loss (EPL) is a psychologically devastating condition affecting the quality of life (QOL). Thus, it needs to be managed by a mind body integrated approach like yoga.The prospective single arm exploratory studyincluded male partners of couples experiencing recurrent pregnancy loss (RPL, n = 30), and recurrent implantation failure (RIF, n = 30) and semen samples wereassessed at the beginning and completion of yoga (6 weeks) (WHO 2010).A significant increase in the sperm concentration, motility, decrease in seminal ROS, DFI and increase in relative sperm telomere length was found at the end of yoga. The relative expression of genes critical for early embryonic developmentnormalized towards the levels of controls. WHOQOL-BREF questionnaire scores to assess QOL also showed improvement.Integration of regular practice yoga into our lifestyle may help in improving seminal redox status, genomic integrity, telomere length, normalizing gene expression and QOL, highlighting the need to use an integrated, holistic approach in management of such cases. This is pertinent for decreasing the transmission of mutation and epimutation load to the developing embryo, improving pregnancy outcomes and decreasing genetic and epigenetic disease burden in the next generation.

Individual disruption of 12 testis-enriched genes via the CRISPR/Cas9 system does not affect the fertility of male mice.

More than 1200 genes have been shown in the database to be expressed predominantly in the mouse testes. Advances in genome editing technologies such as the CRISPR/Cas9 system have made it possible to create genetically engineered mice more rapidly and efficiently than with conventional methods, which can be utilized to screen genes essential for male fertility by knocking out testis-enriched genes. Finding such genes related to male fertility would not only help us understand the etiology of human infertility but also lead to the development of male contraceptives. In this study, we generated knockout mice for 12 genes (Acrv1, Adgrf3, Atp8b5, Cfap90, Cfap276, Fbxw5, Gm17266, Lrrd1, Mroh7, Nemp1, Spata45, and Trim36) that are expressed predominantly in the testis and examined the appearance and histological morphology of testes, sperm motility, and male fertility. Mating tests revealed that none of these genes is essential for male fertility at least individually. Notably, knockout mice for Gm17266 showed smaller testis size than the wild-type but did not exhibit reduced male fertility. Since 12 genes were not individually essential for male fertilization, it is unlikely that these genes could be the cause of infertility or contraceptive targets. It is better to focus on other essential genes because complementary genes to these 12 genes may exist.

CircCamsap1 is dispensable for male fertility in mice.

Background: Circular RNAs (circRNAs) are a large class of RNAs present in mammals. Among these, circCamsap1 is a well-acknowledged circRNA with significant implications, particularly in the development and progression of diverse tumors. However, the potential consequences of circCamsap1 depletion in vivo on male reproduction are yet to be thoroughly investigated. Methods: The presence of circCamsap1 in the mouse testes was confirmed, and gene expression analysis was performed using reverse transcription quantitative polymerase chain reaction. CircCamsap1 knockout mice were generated utilizing the CRISPR/Cas9 system. Phenotypic analysis of both the testes and epididymis was conducted using histological and immunofluorescence staining. Additionally, fertility and sperm motility were assessed. Results: Here, we successfully established a circCamsap1 knockout mouse model without affecting the expression of parental gene. Surprisingly, male mice lacking circCamsap1 (circCamsap1-/-) exhibited normal fertility, with no discernible differences in testicular and epididymal histology, spermatogenesis, sperm counts or sperm motility compared to circCamsap1+/+ mice. These findings suggest that circCamsap1 may not play an essential role in physiological spermatogenesis. Nonetheless, this result also underscores the complexity of circRNA function in male reproductive biology. Therefore, further research is necessary to elucidate the precise roles of other circRNAs in regulating male fertility.

Hypogonadotropic hypogonadism in male tilapia lacking a functional rln3b gene.

Relaxin 3 is a neuropeptide that plays a crucial role in reproductive functions of mammals. Previous studies have confirmed that rln3a plays an important role in the male reproduction of tilapia. To further understand the significance of its paralogous gene rln3b in male fertility, we generated a homozygous mutant line of rln3b in Nile tilapia. Our findings indicated that rln3b mutation delayed spermatogenesis and led to abnormal testes structure. Knocking out rln3b gene resulted in a decrease in sperm count, sperm motility and male fish fertility. TUNEL detection revealed a small amount of apoptosis in the testes of rln3b-/- male fish at 390 days after hatching (dah). RT-qPCR analysis demonstrated that mutation of rln3b gene caused a significant downregulation of steroid synthesis-related genes such as cyp17a1, cyp11b2, germ cell marker gene, Vasa, and gonadal somatic cell marker genes of amh and amhr2. Furthermore, we found a significant down-regulation of hypothalamic-pituitary-gonadal (HPG) axis-related genes, while a significantly up-regulation of the dopamine synthetase gene in the rln3b-/- male fish. Taken together, our data strongly suggested that Rln3b played a crucial role in the fertility of XY tilapia by regulating HPG axis genes.

CCDC28A deficiency causes sperm head defects, reduced sperm motility and male infertility in mice.

Mature spermatozoa with normal morphology and motility are essential for male reproduction. The epididymis has an important role in the proper maturation and function of spermatozoa for fertilization. However, factors related to the processes involved in spermatozoa modifications are still unclear. Here we demonstrated that CCDC28A, a member of the CCDC family proteins, is highly expressed in testes and the CCDC28A deletion leads to male infertility. We found CCDC28A deletion had a mild effect on spermatogenesis. And epididymal sperm collected from Ccdc28a-/- mice showed bent sperm heads, acrosomal defects, reduced motility and decreased in vitro fertilization competence whereas their axoneme, outer dense fibers, and fibrous sheath were all normal. Furthermore, we found that CCDC28A interacted with sperm acrosome membrane-associated protein 1 (SPACA1) and glycogen synthase kinase 3a (GSK3A), and deficiencies in both proteins in mice led to bent heads and abnormal acrosomes, respectively. Altogether, our results reveal the essential role of CCDC28A in regulating sperm morphology and motility and suggesting a potential marker for male infertility.

The cAMP signaling module regulates sperm motility in the liverwort Marchantia polymorpha.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a universal signaling molecule that acts as a second messenger in various organisms. It is well established that cAMP plays essential roles across the tree of life, although the function of cAMP in land plants has long been debated. We previously identified the enzyme with both adenylyl cyclase (AC) and cAMP phosphodiesterase (PDE) activity as the cAMP-synthesis/hydrolysis enzyme COMBINED AC with PDE (CAPE) in the liverwort Marchantia polymorpha. CAPE is conserved in streptophytes that reproduce with motile sperm; however, the precise function of CAPE is not yet known. In this study, we demonstrate that the loss of function of CAPE in M. polymorpha led to male infertility due to impaired sperm flagellar motility. We also found that two genes encoding the regulatory subunits of cAMP-dependent protein kinase (PKA-R) were also involved in sperm motility. Based on these findings, it is evident that CAPE and PKA-Rs act as a cAMP signaling module that regulates sperm motility in M. polymorpha. Therefore, our results have shed light on the function of cAMP signaling and sperm motility regulators in land plants. This study suggests that cAMP signaling plays a common role in plant and animal sperm motility.

Elucidating the Role of OXPHOS Variants in Asthenozoospermia: Insights from Whole Genome Sequencing and an In Silico Analysis.

Infertility is a global health challenge that affects an estimated 72.4 million people worldwide. Between 30 and 50% of these cases involve male factors, showcasing the complex nature of male infertility, which can be attributed to both environmental and genetic determinants. Asthenozoospermia, a condition characterized by reduced sperm motility, stands out as a significant contributor to male infertility. This study explores the involvement of the mitochondrial oxidative phosphorylation (OXPHOS) system, crucial for ATP production and sperm motility, in asthenozoospermia. Through whole-genome sequencing and in silico analysis, our aim was to identify and characterize OXPHOS gene variants specific to individuals with asthenozoospermia. Our analysis identified 680,099 unique variants, with 309 located within OXPHOS genes. Nine of these variants were prioritized due to their significant implications, such as potential associations with diseases, effects on gene expression, protein function, etc. Interestingly, none of these variants had been previously associated with male infertility, opening up new avenues for research. Thus, through our comprehensive approach, we provide valuable insights into the genetic factors that influence sperm motility, laying the foundation for future research in the field of male infertility.

Genetic mutation of Cep76 results in male infertility due to abnormal sperm tail composition.

The transition zone is a specialised gate at the base of cilia/flagella, which separates the ciliary compartment from the cytoplasm and strictly regulates protein entry. We identified a potential new regulator of the male germ cell transition zone, CEP76. We demonstrated that CEP76 was involved in the selective entry and incorporation of key proteins required for sperm function and fertility into the ciliary compartment and ultimately the sperm tail. In the mutant, sperm tails were shorter and immotile as a consequence of deficits in essential sperm motility proteins including DNAH2 and AKAP4, which accumulated at the sperm neck in the mutant. Severe annulus, fibrous sheath, and outer dense fibre abnormalities were also detected in sperm lacking CEP76. Finally, we identified that CEP76 dictates annulus positioning and structure. This study suggests CEP76 as a male germ cell transition zone protein and adds further evidence to the hypothesis that the spermatid transition zone and annulus are part of the same functional structure.

Investigation of sperm hsa-mir-145-5p and MLH1 expressions, seminal oxidative stress and sperm DNA fragmentation in varicocele.

BACKGROUND: Mechanisms by which varicocele causes infertility are not clear and few studies have reported that some miRNAs show expression alterations in men with varicocele. Recently, sperm promoter methylation of MLH1 has been shown to be higher in men diagnosed with varicocele. This study aimed to assess the potential effects of miR-145, which was determined to target MLH1 mRNA in silico on sperm quality and function in varicocele. METHODS: Sperm miR-145 and MLH1 expressions of six infertile men with varicocele (Group 1), nine idiopathic infertile men (Group 2), and nine fertile men (control group) were analyzed by quantitative PCR. Sperm DNA fragmentation was evaluated by TUNEL and the levels of seminal oxidative damage and total antioxidant capacity were analyzed by ELISA. RESULTS: Our results have shown that sperm expression of miR-145 was decreased in Group 1 compared to Group 2 (P = 0.029). MLH1 expression was significantly higher in Group 2 than the controls (P = 0.048). Total antioxidant level and sperm DNA fragmentations of Group 1 and Group 2 were decreased (P = 0.001 and P = 0.011, respectively). Total antioxidant capacity was positively correlated with sperm concentration (ρ = 0.475, P = 0.019), total sperm count (ρ = 0.427, P = 0.037), motility (ρ = 0.716, P < 0.0001) and normal morphological forms (ρ = 0.613, P = 0.001) and negatively correlated with the seminal oxidative damage (ρ=-0.829, P = 0.042) in varicocele patients. CONCLUSION: This is the first study investigating the expressions of sperm miR-145 and MLH1 in varicocele patients. Further studies are needed to clarify the potential effect of miR-145 on male fertility.

A Pilot Analysis of Whole Transcriptome of Human Cryopreserved Sperm.

Sperm cryopreservation is a procedure widely used to store gametes for later use, to preserve fertility in patients prior to gonadotoxic treatments or surgery, and for sperm donation programs. The purpose of the study was to assess the impact of cryopreservation on human sperm transcriptome. Semen samples were collected from 13 normospermic men. Each sample was divided into two aliquots. The total RNA was immediately extracted from one aliquot. The second aliquot was frozen and total RNA was extracted after a week of storage in liquid nitrogen. The RNA samples were randomized in four pools, each of six donors, and analyzed by microarrays. The paired Significance Analysis of Microarray was performed. We found 219 lower abundant transcripts and 28 higher abundant transcripts in cryopreserved sperm than fresh sperm. The gene ontology analysis disclosed that cryopreservation alters transcripts of pathways important for fertility (i.e., spermatogenesis, sperm motility, mitochondria function, fertilization, calcium homeostasis, cell differentiation, and early embryo development), although the increase of some transcripts involved in immune response can compensate for the harmful effects of freezing.

Effects of Slow Freezing and Vitrification of Human Semen on Post-Thaw Semen Quality and miRNA Expression.

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples.

Estimation of genetic parameters for bull conception rate and its genetic correlations with semen production traits in Japanese Black bulls.

The P of achieving pregnancy is an important trait of bull fertility in beef cattle and is defined as the bull conception rate (BCR). This study aimed to clarify and better understand the genetic architecture of the BCR calculated using artificial insemination and pregnancy diagnosis records from a progeny testing program in Japanese Black bulls. In this study, we estimated the genetic parameters of the BCR and their correlation with semen production traits. In addition, we assessed the correlated responses in BCR by considering the selection of semen production traits. Nine hundred and sixteen Japanese Black bulls were selected based on fertility, with 28 869 pregnancy diagnostic records from the progeny testing program. Our results showed that the heritability estimate was 0.04 in the BCR at the first service and 0.14 in BCR for the three services, and an increase in the inbreeding coefficient led to a significant decrease in BCR. The phenotypic trend of BCR remained almost constant over the years, whereas the genetic trend increased. In addition, the changes in the progeny testing year effect showed a similar tendency to the phenotypic trends, suggesting that the phenotypic trends could be mainly due to non-genetic effects, including progeny testing year effects. The estimated genetic correlation of BCR with sperm motility traits was favorably moderate to high (ranging from 0.49 to 0.97), and those with sperm quantity traits such as semen volume were favorably low to moderate (ranging from 0.23 to 0.51). In addition, the correlated responses in BCR at the first service by selection for sperm motility traits resulted in a higher genetic gain than direct selection. This study provides new insights into the genetic factors affecting BCR and the possibility of implementing genetic selection to improve BCR by selecting sperm motility traits in Japanese Black bulls.

Forkhead-associated phosphopeptide binding domain 1 (FHAD1) deficiency impaired murine sperm motility.

Background: Genetic knockout-based studies conducted in mice provide a powerful means of assessing the significance of a gene for fertility. Forkhead-associated phosphopeptide binding domain 1 (FHAD1) contains a conserved FHA domain, that is present in many proteins with phospho-threonine reader activity. How FHAD1 functions in male fertility, however, remains uncertain. Methods: Fhad1-/- mice were generated by CRISPR/Cas9-mediated knockout, after which qPCR was used to evaluate changes in gene expression, with subsequent analyses of spermatogenesis and fertility. The testis phenotypes were also examined using immunofluorescence and histological staining, while sperm concentrations and motility were quantified via computer-aided sperm analysis. Cellular apoptosis was assessed using a TUNEL staining assay. Results: The Fhad1-/-mice did not exhibit any abnormal changes in fertility or testicular morphology compared to wild-type littermates. Histological analyses confirmed that the testicular morphology of both Fhad1-/-and Fhad1+/+ mice was normal, with both exhibiting intact seminiferous tubules. Relative to Fhad1+/+ mice, however, Fhad1-/-did exhibit reductions in the total and progressive motility of epididymal sperm. Analyses of meiotic division in Fhad1-/-mice also revealed higher levels of apoptotic death during the first wave of spermatogenesis. Discussion: The findings suggest that FHAD1 is involved in both meiosis and the modulation of sperm motility.

Seminal cell-free DNA as a potential marker for in vitro fertility of Nellore bulls.

PURPOSE: This study aimed to identify a marker for freezability and in vitro fertility of sperm samples before freezing. METHODS: Semen was collected from nine Nelore bulls; half of the ejaculate was used for seminal plasma cell-free DNA (cfDNA) quantification, and the other half was cryopreserved. Evaluation of sperm movement using computer-assisted semen analysis and plasma membrane integrity and stability, acrosomal integrity, apoptosis, and mitochondrial potential using flow cytometry were performed on fresh and frozen/thawed semen at 0, 3, 6, and 12 h after thawing. Frozen/thawed sperm was also used for in vitro embryo production. cfDNA was extracted from each bull, and the total DNA and number of cell-free mitochondrial DNA (cfmtDNA) copies were quantified. Semen from each animal was used for IVF, and cleavage, blastocyst formation, and cell counts were evaluated. RESULTS: Two groups were formed and compared based on the concentrations of cfDNA and cfmDNA present: low-cfDNA and high-cfDNA and low-cfmtDNA and high-cfmtDNA. Up to 12 h post-thawing, there were no differences between the groups in the majority of the sperm parameters evaluated. Cleavage, day 6 and 7 blastocyst rates, and the number of cells were higher in the high cfDNA group than in the low cfDNA group. Similar results were observed for cfmtDNA, except for the number of cells, which was similar between the groups. CONCLUSION: The concentration of cfDNA and the relative number of copies of cfmtDNA in seminal plasma cannot predict the freezability of semen but can be used to predict in vitro embryo production.

Genome-Wide Association Analysis of Semen Characteristics in Piétrain Boars.

Since artificial insemination is common practice in pig breeding, the quality and persistence of the semen are decisive for the usability of individual boars. In the current study, genome-wide association analyses were performed to investigate the genetic variability underlying phenotypic variations in semen characteristics. These traits comprise sperm morphology and sperm motility under different temporal and thermal storage conditions, in addition to standard semen quality parameters. Two consecutive samples of the fourth and fifth ejaculates from the same boar were comprehensively analyzed in a genotyped Piétrain boar population. A total of 13 genomic regions on different chromosomes were identified that contain single-nucleotide polymorphisms significantly associated with these traits. Subsequent analysis of the genomic regions revealed candidate genes described to be involved in spermatogenesis, such as FOXL3, GPER1, PDGFA, PRKAR1B, SNRK, SUN1, and TSPO, and sperm motility, including ARRDC4, CEP78, DNAAF5, and GPER1. Some of these genes were also associated with male fertility or infertility in mammals (e.g., CEP78, GPER1). The analyses based on these laboriously determined and valuable phenotypes contribute to a better understanding of the genetic background of male fertility traits in pigs and could prospectively contribute to the improvement of sperm quality through breeding approaches.

CCDC157 is essential for sperm differentiation and shows oligoasthenoteratozoospermia-related mutations in men.

Oligoasthenoteratospermia (OAT), characterized by abnormally low sperm count, poor sperm motility, and abnormally high number of deformed spermatozoa, is an important cause of male infertility. Its genetic basis in many affected individuals remains unknown. Here, we found that CCDC157 variants are associated with OAT. In two cohorts, a 21-bp (g.30768132_30768152del21) and/or 24-bp (g.30772543_30772566del24) deletion of CCDC157 were identified in five sporadic OAT patients, and 2 cases within one pedigree. In a mouse model, loss of Ccdc157 led to male sterility with OAT-like phenotypes. Electron microscopy revealed misstructured acrosome and abnormal head-tail coupling apparatus in the sperm of Ccdc157-null mice. Comparative transcriptome analysis showed that the Ccdc157 mutation alters the expressions of genes involved in cell migration/motility and Golgi components. Abnormal Golgi apparatus and decreased expressions of genes involved in acrosome formation and lipid metabolism were detected in Ccdc157-deprived mouse germ cells. Interestingly, we attempted to treat infertile patients and Ccdc157 mutant mice with a Chinese medicine, Huangjin Zanyu, which improved the fertility in one patient and most mice that carried the heterozygous mutation in CCDC157. Healthy offspring were produced. Our study reveals CCDC157 is essential for sperm maturation and may serve as a marker for diagnosis of OAT.

Target silencing of porcine SPAG6 and PPP1CC by shRNA attenuated sperm motility.

The quality of sperm significantly influences the reproductive efficiency of pig herds. High-quality sperm is necessary for efficient fertilization and to maximize the litter numbers in commercial pig farming. However, the understanding of genes regulating porcine sperm motility and viability is limited. In this study, we validated porcine sperm/Sertoli-specific promoters through the luciferase reporter system and identified vital genes for sperm quality via loss-of-function means. Further, the shRNAs driven by the ACE and SP-10 promoters were used to knockdown the SPAG6 and PPP1CC genes which were provisionally important for sperm quality. We assessed the effects of SPAG6 and PPP1CC knockdown on sperm motility by using the sperm quality analyzer and flow cytometry. The results showed that the ACE promoter is active in both porcine Sertoli cells and sperms, whereas the SP-10 promoter is operating exclusively in sperm cells. Targeted interference with SPAG6 and PPP1CC expression in sperm cells decreases the motility and increases apoptosis rates in porcine sperms. These findings not only offer new genetic tools for targeting male germ cells but also highlight the crucial roles of SPAG6 and PPP1CC in porcine sperm function.