(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . Golgi associated RAB2 interactor protein family contributes to murine male fertility to various extents by assuring correct morphogenesis of sperm heads [1] ['Haoting Wang', 'Graduate School Of Pharmaceutical Sciences', 'Osaka University', 'Suita', 'Osaka', 'Research Institute For Microbial Diseases', 'Rie Iida-Norita', 'Daisuke Mashiko', 'Anh Hoang Pham', 'Haruhiko Miyata'] Date: 2024-07 Sperm heads contain not only the nucleus but also the acrosome which is a distinctive cap-like structure located anterior to the nucleus and is derived from the Golgi apparatus. The Golgi Associated RAB2 Interactors (GARINs; also known as FAM71) protein family shows predominant expression in the testis and all possess a RAB2-binding domain which confers binding affinity to RAB2, a small GTPase that is responsible for membrane transport and vesicle trafficking. Our previous study showed that GARIN1A and GARIN1B are important for acrosome biogenesis and that GARIN1B is indispensable for male fertility in mice. Here, we generated KO mice of other Garins, namely Garin2, Garin3, Garin4, Garin5a, and Garin5b (Garin2-5b). Using computer-assisted morphological analysis, we found that the loss of each Garin2-5b resulted in aberrant sperm head morphogenesis. While the fertilities of Garin2 -/- and Garin4 -/- males are normal, Garin5a -/- and Garin5b -/- males are subfertile, and Garin3 -/- males are infertile. Further analysis revealed that Garin3 -/- males exhibited abnormal acrosomal morphology, but not as severely as Garin1b -/- males; instead, the amounts of membrane proteins, particularly ADAM family proteins, decreased in Garin3 KO spermatozoa. Moreover, only Garin4 KO mice exhibit vacuoles in the sperm head. These results indicate that GARINs assure correct head morphogenesis and some members of the GARIN family function distinctively in male fertility. Approximately 5% of males exhibit infertility, which has become a significant problem worldwide. One major reason for male infertility is teratozoospermia, characterized by spermatozoa with abnormal morphology. However, the genetic cause of teratozoospermia is largely unknown. In our previous study, we found two proteins belonging to the Golgi associated RAB2 interactor (GARIN) protein family are responsible for sperm acrosome biogenesis and mutation of each one of them causes globozoospermia in mice, which is a subset of teratozoospermia defined as spermatozoa that show round sperm heads with abnormal acrosome morphology. In this study, we generated knockout mice of each gene belonging to the Garin family. By applying elliptic Fourier descriptors (EFDs) and principal component (PC) analysis on sperm heads, we demonstrated that GARINs are all responsible for correct sperm head morphogenesis. GARINs are all conserved in humans, and expanding the understanding of GARINs potentially contributes to the elucidation of human male infertility. Funding: This research was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI grants ( https://www.jsps.go.jp/english/e-grants/index.html , JP22H03214, JP23K18328 to H.M. and JP19H05750, JP21H04753, JP21H05033 to M.I.); Takeda Science Foundation grant ( https://www.takeda-sci.or.jp/ ) to H.M. and M.I.; JST FOREST ( https://www.jst.go.jp/souhatsu/en/index.html , JPMJFR211F to H.M.); the Eunice Kennedy Shriver National Institute of Child Health and Human Development ( https://www.nichd.nih.gov/ , P01HD087157 and R01HD088412 to M.I.); and the Bill & Melinda Gates Foundation ( https://www.gatesfoundation.org/ , Grand Challenges Explorations grant INV-001902 to M.I.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In this study, we generated 5 single KO mice of Garin2, Garin3, Garin4, Garin5a, and Garin5b and aimed to elucidate the characteristics of GARINs. we demonstrated that GARIN2, GARIN3, GARIN4, GARIN5A, and GARIN5B are all important for sperm head morphogenesis, along with GARIN3 being vital for acrosome biogenesis and male fertility. Teratozoospermia is a major clinical cause of male infertility, which is defined as over 85% of the spermatozoa carrying abnormal morphology in humans [ 11 , 12 ]. Moreover, globozoospermia, a subset of teratozoospermia, is specifically characterized by spermatozoa with abnormal acrosome morphology [ 11 ]. The genetic and molecular mechanisms underlying teratozoospermia are largely unknown, and the mouse knockout (KO) model is used as an outstanding animal model to elucidate the genetic cause. In our previous study, we identified two globozoospermia-related genes: Golgi Associated RAB2 Interactor 1A (Garin1a; Fam71f2) and Garin1b (Fam71f1) [ 13 ]. Garin1a and Garin1b are predominantly expressed in the testes and KO of Garin1a and Garin1b causes subfertility and infertility in males, respectively, due to abnormal acrosomal morphology [ 13 ]. Furthermore, the same study showed that GARIN1B binds to both RAB2A and RAB2B which are small GTPases with high amino acid sequence similarities and are involved in vesicle trafficking. In contrast, the functions of other GARIN paralogues, namely GARIN3, GARIN4, GARIN5A, and GARIN5B, in male fertility are not clear although it has been shown recently that Garin2 (Fam71d) is not essential for male fertility in mice [ 14 ]. The acrosome, a distinctive Golgi-derived structure situated at the anterior aspect of the sperm head, plays an important role in sperm-egg interaction [ 1 – 3 ]. The acrosome reaction, characterized as an exocytosis event releasing the enzymes stored within the acrosome, assists sperm penetration through the zona pellucida (ZP) of oocytes [ 1 , 2 , 4 ]. Simultaneously, the acrosome reaction assists the migration of receptors that facilitate sperm-egg fusion [ 5 – 7 ]. Spermiogenesis can be divided into 4 phases based on the morphology of the acrosome and nucleus: Golgi phase, cap phase, acrosome phase, and maturation phase [ 8 ]. Starting from the Golgi phase, the Golgi apparatus actively produces vesicles containing various glycoproteins, and then, multiple vesicles fuse to form a larger vesicle which is called an acrosomal vesicle near the nucleus [ 9 ]. In the cap phase, acrosomal vesicles spread over the surface and become flattened around the nucleus. After the cap phase, the skirt-like microtubule-based structure, the manchette is formed, which facilitates nuclear elongation during the acrosome phase [ 10 ]. The nucleus and acrosomal vesicle then further elongate to complete spermiogenesis in the maturation phase. Spermatozoa are generated through a specialized biological process known as spermatogenesis. This intricate process occurs in seminiferous tubules of the testis, comprising a series of discrete steps. At the first step, spermatogonia undergo mitosis, transitioning into spermatocytes. Meiosis then occurs to transform diploid spermatocytes into haploid round spermatids. Spermiogenesis follows meiosis and is characterized by sixteen steps in mice. Spermiogenesis is a complicated process that involves nuclear condensation, morphogenesis of the sperm head, and formation of the sperm tail. These events collectively ensure the integrity of spermatozoa and secure the correct interaction of spermatozoa with oocytes. Results Garin2, Garin3, Garin5a, and Garin5b KO mice exhibit impaired sperm motility We next examined sperm motility by computer-assisted sperm analysis (CASA) after 10 and 120 minutes incubation in a capacitation medium as defective motility could result in impaired ZP penetration [19]. Using the same CASA system, minor motility defects were found in spermatozoa with impaired ZP penetration [20]. For both 10 and 120 minutes, the percentages of motile spermatozoa were significantly decreased only in Garin2 KO spermatozoa compared to WT spermatozoa (S4A and S4B Fig). Despite the percentages of motile spermatozoa being comparable in other Garin KO mice compared to WT mice, kinetic parameters such as straight line velocity (VSL), curvilinear velocity (VCL), and average path velocity (VAP) were reduced in Garin3, Garin5a, and Garin5b KO mice after 10 minutes of incubation (Fig 3D–3F). After 120 minutes of incubation, VSL was lower in Garin5b KO spermatozoa, VCL was lower in Garin2, Garin3, and Garin5b KO spermatozoa, and VAP was lower in Garin2 and Garin5b KO spermatozoa (Fig 3G–3I). Although Garin2, Garin3, Garin5a, and Garin5b KO spermatozoa exhibited impaired motility, kinetic parameters of Garin5b KO spermatozoa were still comparable with or higher than those of inbred C57BL/6J WT spermatozoa (S4C and S4D Fig), suggesting that the impaired ZP penetration cannot be explained only by diminished sperm motility. Garin3 KO spermatozoa display abnormal acrosomal morphology We have been shown that each KO of Garin1a and Garin1b results in abnormal sperm head and acrosomal morphology [13]. Therefore, we observed the acrosome of Garin2-5b KO spermatozoa with PNA staining. Although the majority of the acrosome from Garin2-5b KO spermatozoa seemed normal, some of the acrosomes of Garin3 KO spermatozoa appeared to expand (Fig 5A), consistent with Garin1a and Garin1b KO spermatozoa [13]. We quantified the PNA-positive area in WT and Garin2-5b KO spermatozoa and found that the acrosome area of Garin3 KO spermatozoa was significantly larger than that of WT spermatozoa (Fig 5B), suggesting that abnormal head morphology of Garin3 KO spermatozoa may be caused by impaired acrosome biogenesis. In addition, we observed nuclear vacuoles in the Hoechst staining of the Garin4 KO spermatozoa (Fig 5A), consistent with the phase contrast observation (Fig 4A). PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 5. Acrosome morphology of Garin2-5b KO spermatozoa and acrosome reaction of Garin3 KO spermatozoa. (A) Acrosomal morphology of WT and Garin2-5b (Garin2, 3, 4, 5a, and 5b) KO mice. The acrosome was stained with PNA (red) while nuclei were stained with Hoechst33342 (Blue). (B) PNA positive area of WT and Garin2-5b KO spermatozoa were quantified. Garin2-5b KO were compared with WT (One-way ANOVA, a, P < 0.05). For each KO mouse line and WT mouse, 3 males were analyzed. (C) Acrosome reaction rates of WT and Garin3 KO spermatozoa after incubating for 10 minutes, 4 hours, and after adding Ca2+ ionophore A23187. The difference between acrosome reaction rates of WT and Garin3 KO mice in each condition was not significant (Student’s t-test, P > 0.05). https://doi.org/10.1371/journal.pgen.1011337.g005 Because acrosomal morphology was abnormal in Garin3 KO spermatozoa, we measured acrosome reaction rates after 10 minutes and 4 hours incubation in a capacitation medium, and also after treatment with the Ca2+ ionophore A23187 [23]. There were no significant differences in acrosome reaction rates between WT and Garin3 KO spermatozoa in these conditions (Fig 5C), supporting the idea that the ZP penetration failure is caused by abnormal sperm head morphology but not impaired acrosome reaction. Deletion of Garin3 affects sperm head plasma membrane proteins To further analyze the effects of Garin3 deletion, which caused the severest impairment in male fertility among Garin2-5b KOs, we performed mass spectrometry (MS) analyses of mature spermatozoa collected from cauda epididymis. We found that not only GARIN3 was completely absent in Garin3 KO spermatozoa, but also 18 proteins such as ADAM4, ADAM6A, and ADAM6B were significantly downregulated in Garin3 KO spermatozoa (S2 Table). We performed gene ontology (GO) analysis on downregulated proteins (Fig 6A) and found that proteins categorized in the sperm head plasma membrane and male gonad development were significantly downregulated in Garin3 KO spermatozoa. It has been shown that the expression levels of ADAM4 and ADAM6 are dramatically reduced in Adam2 KO and Adam3 KO spermatozoa, and ADAM6 forms a complex with ADAM2 and ADAM3 [24]. Therefore, we analyzed the amounts of ADAM proteins for those antibodies that were available for us and found that ADAM2, ADAM3, and ADAM32 were downregulated in Garin3 KO spermatozoa (Fig 6B). As a negative control, the amount of IZUMO1, a transmembrane protein localized in the acrosomal membrane, was not affected. These results suggest that not only head morphology but also the amounts of plasma membrane proteins were affected in Garin3 KO spermatozoa. Moreover, in Garin2-5b KO spermatozoa, only Garin3 KO spermatozoa showed downregulated ADAM3 (S6A Fig), suggesting the functional differences of GARIN3 among GARIN2-5B. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 6. KO of Garin3 led to the loss of ADAMs and aberrant ZP-binding ability. (A) GO analysis of proteins significantly downregulated in Garin3 KO spermatozoa (S2 and S6 Tables). MF, CC, and BP are abbreviations for molecular function, cellular component, and biological process, respectively. (B) Western blotting analyses revealed the amounts of ADAM2, ADAM3, and ADAM32 decreased in Garin3 KO spermatozoa. IZUMO1 and ACTB were detected as endogenous controls. (C) Representative image of ZP-binding analyses of WT and Garin3 KO spermatozoa. (D) Numbers of ZP-bound spermatozoa per egg. Garin3 KO spermatozoa exhibited lower ZP-binding ability compared to WT spermatozoa. For Garin3-/- and WT mice, 3 males were used for the ZP-binding assay (Student’s t-test, c, P < 0.001). https://doi.org/10.1371/journal.pgen.1011337.g006 Because it has been shown that in the absence of ADAM2, ADAM3, or ADAM6, the ZP binding ability is severely impaired [25–27], we analyzed ZP binding ability of Garin3 KO spermatozoa (Fig 6C). Although the ZP binding ability of Garin3 KO spermatozoa is significantly reduced, 5.5 spermatozoa on average could still bind to the ZP, which is about 31.3% of the binding ability compared to WT spermatozoa (Fig 6C and 6D). These results suggest that the combinatorial effect of impaired sperm head morphology and ZP binding ability resulted in ZP penetration failure in Garin3 KO spermatozoa. [END] --- [1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011337 Published and (C) by PLOS One Content appears here under this condition or license: Creative Commons - Attribution BY 4.0. via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/