Acephalic spermatozoa syndrome (ASS) is a rare and severe form of male infertility that poses significant challenges for affected individuals and their families[3–9]. It is often associated with genetic factors. In recent years, researchers have made progress in identifying specific genes that play a role in ASS. Mutations in several genes, such as PMFBP1, SUN5, TSGA10, BRDT, HOOK1, DNAH6, ACTRT1, SPATC1L and SPATA20, have been identified as causative factors for some patients[13–25]. However, it is important to note that these mutations only account for a portion of individuals diagnosed with ASS. To gain further insights into the pathogenesis of this condition and potentially identify additional contributing factors or genetic variations involved in ASS development, more extensive studies are needed. In our study mentioned above, we encountered a primary infertile patient who was diagnosed with ASS through semen analysis and Papanicolaou staining techniques. By utilizing Sanger sequencing methods on the patient's DNA sample, we identified a homozygous splice-site mutation (2089-1G > T; NM_031293.2) within the PMFBP1 gene as the likely pathogenic factor responsible for causing ASS in this particular patient.
According to previous studies, PMFBP1 and SUN5 mutations are primarily responsible for acephalospermia [11, 14–17, 20]. The PMFBP1 gene is located on human chromosome 16 and consists of 27 exons. The coding region of the gene spans 3,024 bases and encodes a protein consisting of 1,007 amino acids[26]. PMFBP1 is specifically expressed in both human and mouse testes [14, 27]. It localizes to the junction area between the head and flagella of sperm cells, and its expression is lost in patients carrying PMFBP1 mutations [11, 14]. Functionally, PMFBP1 interacts with SUN5 and SPATA6 to facilitate the connection between the head and tail of sperm cells. In male mice, mutations in PMFBP1 disrupt this cooperation, leading to headless sperm. Therefore, it is believed that PMFBP1 mutation plays a significant role in acephalospermia, which can cause male infertility [11, 14]. The separation in subtype II is positioned between the nucleus and the proximal centriole[28]. Reproductive success was observed in clinical pregnancies achieved through ICSI methods in individuals with subtype II mutations within PMFBP1 [27, 28].
In our study, we analyzed the protein expression of PMFBP1 in patient samples compared to controls via western blotting. Our results revealed an absence of detectable levels of the PMFBP1 protein in patient sperm, while control samples exhibited positive expression. These findings indicate that splice site mutation leads to an absence of functional PMFBP1 protein production. Immunofluorescence analysis further confirmed that splice site mutation resulted in the absence of detectable levels of the PMFPB11 protein.
Furthermore, to further investigate the functional consequences of the splice site mutation in PMFBP1, we constructed minigenes. These minigenes were then transfected into HEK-293T cells. The results of our experiments revealed that this particular splice-site mutation led to a deletion of 4 base pairs in exon 15 when tested in vitro. This deletion ultimately resulted in an ASS phenotype, which is characterized by certain developmental abnormalities. Based on our observations, we hypothesized that this specific mutation introduces a premature stop codon after the translation of six amino acids (NM_031293.2, c.2089-1G > T, p.I697Pfs7*). It is important to note that such mutations can have significant implications for protein structure and function. To confirm these findings and gain further insights into the effects of this mutation on mRNA splicing and protein production, we attempted RT‒PCR analysis using sperm mRNA from the studied patients. However, due to the limited availability of sperm heads from these individuals, we were unable to extract sufficient sperm mRNA for successful analysis. Despite these limitations, our initial experiments with minigenes provided valuable information regarding the impact of this splice site mutation on PMFBP1 function. Further studies utilizing alternative approaches or larger sample sizes may be necessary to fully elucidate its role in disease development and progression.
In conclusion, our study identified a homozygous splice-site PMFBP1 mutation in an ASS patient who leads to the removal of 4 bp in exon 15 and subsequently results in the absence of expression of the PMFBP1 protein. Our findings add to the growing body of evidence supporting the involvement of PMFBP1 in male infertility disorders such as ASS. Understanding how this mutation affects sperm development at both the cellular and molecular levels may provide valuable insights into potential therapeutic targets or interventions aimed at improving fertility outcomes for affected individuals.