PMC9889981_01
Identification and Bioinformation Analysis of HYDIN Mutations in Two unrelated families. (A)Two compound heterozygous mutations (M1-M4) of HYDIN were identified in two subjects with asthenoteratozoospemia. M2 of patient AY078 was inherited from heterozygous parents. Because the bioinformation of P1's father was incapable gained, M1 in AY078 was undetermined whether is inherited from his father or is new. M3 and M4 of patient 2 AY079 were obviously inherited from his heterozygous parents. The mutational positions were indicated with red arrow under Sanger sequencing results below. (B)Schematic representation of HYDIN exons and protein product. The conservation of variant residues among different species were verified by sequence alignment. The positions of variants were indicated in dots lines. Green square represents typical ASH (ASPH-SPD-2-Hydin) domain, blue square stands for Hydin-ADK domain, according to the NCBI browser.
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SPEF2 and another CP marker, SPAG6, were significantly reduced in sperm from AY078 ( Figures 7A, D  and  Figures S1A, D ). STRING analysis further indicated that HYDIN may be highly associated with RSPH4A ( Figure 7B ). We also examined the abundance and location of other components of the radical spoke complexes, RSPH1 and RSPH3, which were significantly reduced, similar to those of RSPH4A ( Figures S1B-D ).
Morphology analysis of sperm from HYDIN-deficient patient AY078. (A, B) Morphology analysis showed various defects in head, neck and flagella of sperm from AY078 via H&E staining and SEM. Comparing with normal-shaped head that nucleus capped by acrosome, long and smooth flagella in control spermatozoa, the amorphous head, pyramid head and abnormal acrosome (orange arrowhead), the short and coiled flagella were observed in P1. Scale bar: 10mum in (A), 2mum in (B).
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AY078. AY078 sperm also displayed various flagellar deformities, including coiled (51.5%), short (21.3%), and angulated flagella (11.4%;  Figure 2A  and  Table 2 ). Similar phenotypes were also observed by SEM: most AY078 spermatozoa displayed abnormal (amorphous and pyramid) head morphology with few normal acrosome forms, as well as a thin bent neck and short coiled flagella ( Figure 2B ).
Ultrastructural deficiency in sperm in HYDIN-deficient AY078 comparing with normal control. (A) Magnification of longitudinal section of sperm showed that the anterior two thirds of nucleus was covered by acrosome, which was packaged with OAM and IAM (orange arrow), and firm linkage between head and tail in normal-shaped sperm (yellow arrow). While, it was showed that various malformations in AY078: uneven nuclear concentration, damaged and exposed acrosomal contents accompanied the outer acrosomal membrane stripped from the nucleus (orange arrow), abnormal or desultory connection between head and flagella (yellow arrow). Scale bar: 2mum. (B) TEM analysis of cross-section ultrastructure within flagella of AY078 and normal sperm. Typical axoneme and peri-axoneme: ODF, MS in mid-piece or FS in principle-piece surrounded the "9+2" structure, that nine MDs and one pair CP, were showed in cross-section of mid-piece, principle-piece and end-piece of sperm flagella from control man. The projection of RS also was captured lightly through TEM in three cross-section of control sperm. It was found that the dramatically reduction of CP and RS complex and destroy on mitochondrial sheath in AY078 flagella. CP, central pair; MD, microtubules doublet; RS, radical spoke; ODF, outer dense fiber; MS, mitochondrial sheath; FS, fibrous sheath. Scale bar: 500 nm.
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Figure 3A , most acrosomes were damaged and stripped from the nuclear envelope, with more than one large nuclear vacuole, a deep depression on the surface of the nucleus, and low nuclear concentration in some severely impaired sperm. Furthermore, the sperm head-tail junction structure was damaged in AY078 sperm, with head and tail separation and a bare, thin structure at the end of the mid-region ( Figure 3A ). Most sperm lacked CP axonemal composition and were characterized by "9+0" and "9+1" axonemes, and with no other obvious abnormalities in the mitochondrial sheath (MS), outer dense fibers (ODF), or doublets of microtubules (DMT) structures were observed ( Figure 3B ).
The IF and RT-qPCR assays in patient AY078 and fertile individuals. (A) IF analysis of HYDIN in sperm from control man and AY078. In the fertile individual, signals of HYDIN localized along the sperm flagella, besides this, were clear found in the neck and anterior head (acrosome). By contrast, the HYDIN staining was dramatically reduced or absent in sperm of P1 harboring HYDIN variants. Scale bar: 10mum. (B) The relative mRNA expression level of HYDIN in AY078 and control individual. The HYDIN mRNA level of HYDIN-deficient subjects was significantly reduced compared with that in the control. ** P < 0.01.
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HYDIN variant, significantly fewer HYDIN signals were distributed on the sperm acrosomal region and neck, while signals on the flagella were comparable to the control ( Figure 4A ). HYDIN mRNA expression levels were significantly reduced in spermatozoa from AY078, indicating that compound heterozygous splicing had a negative influence on HYDIN expression in AY078 ( Figure 4B ).
The distribution and expression of acrosomal associated proteins in AY078 and control individual. (A-C) Immunofluorescence staining assays wereperformed on the sperm of AY078 and normal subject using anti-ACTL7A(red in A), anti-ACROSIN (red in B) and anti-PLCz1 (red in C). Compared with cap-like staining of ACTL7A and ACROSIN, localized on anterior head in normal sperm, the signal in P1 of those proteins were absent from acrosome. The PLCz1 normally localized in cap-like area of acrosome, while was absent, decreased and dispersive from normal region. Anti-ac-tubulin (green) marked the sperm flagella, Hoechst (blue) marked the nucleus of spermatozoa. Scale bars: 10mm. (D) WB assays analysis the expression levels of ALTL7A, ACROSIN and PLCz1 in sperm obtained from P1 and normal control. The results of WB assays were accordance with those of immunofluorescence assays described above. b-actin was used as internal reference.
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ACTL7A, acrosin, and PLCzeta1, using WB and IF. ACTL7A and acrosin signals were almost absent in AY078 spermatozoa, while abnormal localization and significantly reduced signals were observed for PLCzeta1 compared to the normal control (Figures 5A-C). Consistently, immunoblot analysis showed that ACTL7A, acrosin, and PLCzeta1 were absent or dramatically downregulated in spermatozoa from AY078 ( Figure 5D ).
The distribution and expression of proteins related to centrosome and middle piece formation in AY078 and control individual. (A-C) IF results on the sperm of AY078 and normal subject using anti-Centrin1(red in A), anti-TOMM20 (red in B) and anti-SEPT4 (red in C). It was demonstrated that Centrin1 express at centriole, TOMM20 locate at mitochondrial sheath, and SEPT4 distribute on annulus ring in normal sperm. While, it was found that different degrees of reduction of Centrin1, TOMM20 and SEPT4 in sperm from AY078. Anti-ac-tubulin (green) marked the sperm flagella, Hoechst (blue) marked the nucleus of spermatozoa. Scale bars: 5mum in (A), 10mum in (B-D) WB assays analysis the expression levels of Centrin1, TOMM20 and SEPT4 in sperm obtained from AY078 and normal control. The results of WB assays were accordance with those of immunofluorescence assays described above. beta-actin was used as internal reference.
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ASH-containing protein concentrated on the neck, which may be related to centrosome and mid-region formation, we examined the localization and levels of components of the centrosome, mitochondrial sheath, and annulus ring. As shown in  Figure 6 , the numbers of centrosomes, mitochondrial sheaths, and annulus rings were decreased to different degrees, and the levels of Centrin1, TOMM20, and SEPT4 were also decreased in sperm from AY078, indicating that HYDIN deficiency may contribute to these defects.
Deficiency of axoneme and appendages in sperm from AY078. (A, C) The immunofluorescence assays of flagella associated proteins SPEF2 (Sperm Flagellar2) and RSPH4A. Anti- SPEF2 (red in B) and Anti- RSPH4A (red in C) normally localized along the sperm flagella in the control sperm. However, expressions of SPEF2 and RSPH4A were almost absent in sperm obtained from AY078 harboring HYDIN variants. Anti-ac-tubulin (green) marked the sperm flagella. Hoechst (blue) labeled the nucleus of spermatozoa. Scale bars: 10mum (B) STRING analysis towards HYDIN. Evidently, it was indicated that HYDIN may be highly connected with RSPH4A. (D) The expression levels of SPEF2 and RSPH4A in spermatozoa were analyzed by western blot from normal individual and HYDIN-deficient subjects. The results showed obviously decrease of those proteins. beta-actin was used as internal reference.
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SPEF2 and another CP marker, SPAG6, were significantly reduced in sperm from AY078 ( Figures 7A, D  and  Figures S1A, D ). STRING analysis further indicated that HYDIN may be highly associated with RSPH4A ( Figure 7B ). RSPH4A on spermatozoa from AY078. RSPH4A immunostaining was localized along the entire flagella in normal sperm, whereas RSPH4A signals and levels were markedly decreased ( Figures 7C, D ).