HSF5 Deficiency Causes Male Infertility Involving Spermatogenic Arrest at Meiotic Prophase I in Humans and Mice.

Meiosis is a specialized cell division process that generates gametes for sexual reproduction. However, the factors and underlying mechanisms involving meiotic progression remain largely unknown, especially in humans. Here, it is first showed that HSF5 is associated with human spermatogenesis. Patients with a pathogenic variant of HSF5 are completely infertile. Testicular histologic findings in the patients reveal rare postmeiotic germ cells resulting from meiotic prophase I arrest. Hsf5 knockout (KO) mice confirms that the loss of HSF5 causes defects in meiotic recombination, crossover formation, sex chromosome synapsis, and sex chromosome inactivation (MSCI), which may contribute to spermatocyte arrest at the late pachytene stage. Importantly, spermatogenic arrest can be rescued by compensatory HSF5 adeno-associated virus injection into KO mouse testes. Mechanistically, integrated analysis of RNA sequencing and chromatin immunoprecipitation sequencing data revealed that HSF5 predominantly binds to promoters of key genes involved in crossover formation (e.g., HFM1, MSH5 and MLH3), synapsis (e.g., SYCP1, SYCP2 and SYCE3), recombination (TEX15), and MSCI (MDC1) and further regulates their transcription during meiotic progression. Taken together, the study demonstrates that HSF5 modulates the transcriptome to ensure meiotic progression in humans and mice. These findings will aid in genetic diagnosis of and potential treatments for male infertility.

Deficiency of MFSD6L, an acrosome membrane protein, causes oligoasthenoteratozoospermia in humans and mice.

Oligoasthenoteratozoospermia is an important factor affecting male fertility and has been found to be associated with genetic factors. However, there are still a proportion of oligoasthenoteratozoospermia cases that cannot be explained by known pathogenic genetic variants. Here, we perform genetic analyses and identify bi-allelic loss-of-function variants of MFSD6L from an oligoasthenoteratozoospermia affected family. Mfsd6l knock-out male mice also present male subfertility with reduced sperm concentration, motility, and deformed acrosomes. Further mechanistic analyses reveal that MFSD6L, as an acrosome membrane protein, plays an important role in the formation of acrosome by interacting with inner acrosomal membrane protein SPACA1. Moreover, poor embryonic development is consistently observed after intracytoplasmic sperm injection treatment using spermatozoa from MFSD6L-deficient man and male mice. Collectively, our findings reveal that MFSD6L is required for the anchoring of sperm acrosome and head shaping. The deficiency of MFSD6L affects male fertility and causes oligoasthenoteratozoospermia in humans and mice.

Gene-knockout by iSTOP enables rapid reproductive disease modeling and phenotyping in germ cells of the founder generation.

Cytosine base editing achieves C•G-to-T•A substitutions and can convert four codons (CAA/CAG/CGA/TGG) into STOP-codons (induction of STOP-codons, iSTOP) to knock out genes with reduced mosaicism. iSTOP enables direct phenotyping in founders' somatic cells, but it remains unknown whether this works in founders' germ cells so as to rapidly reveal novel genes for fertility. Here, we initially establish that iSTOP in mouse zygotes enables functional characterization of known genes in founders' germ cells: Cfap43-iSTOP male founders manifest expected sperm features resembling human "multiple morphological abnormalities of the flagella" syndrome (i.e., MMAF-like features), while oocytes of Zp3-iSTOP female founders have no zona pellucida. We further illustrate iSTOP's utility for dissecting the functions of unknown genes with Ccdc183, observing MMAF-like features and male infertility in Ccdc183-iSTOP founders, phenotypes concordant with those of Ccdc183-KO offspring. We ultimately establish that CCDC183 is essential for sperm morphogenesis through regulating the assembly of outer dynein arms and participating in the intra-flagellar transport. Our study demonstrates iSTOP as an efficient tool for direct reproductive disease modeling and phenotyping in germ cells of the founder generation, and rapidly reveals the essentiality of Ccdc183 in fertility, thus providing a time-saving approach for validating genetic defects (like nonsense mutations) for human infertility.

Coiled-coil domain-containing 38 is required for acrosome biogenesis and fibrous sheath assembly in mice.

During spermiogenesis, haploid spermatids undergo dramatic morphological changes to form slender sperm flagella and cap-like acrosomes, which are required for successful fertilization. Severe deformities in flagella cause a male infertility syndrome, multiple morphological abnormalities of the flagella (MMAF), while acrosomal hypoplasia in some cases leads to sub-optimal embryonic developmental potential. However, evidence regarding the occurrence of acrosomal hypoplasia in MMAF is limited. Here, we report the generation of base-edited mice knocked out for coiled-coil domain-containing 38 (Ccdc38) via inducing a nonsense mutation and find that the males are infertile. The Ccdc38-KO sperm display acrosomal hypoplasia and typical MMAF phenotypes. We find that the acrosomal membrane is loosely anchored to the nucleus and fibrous sheaths are disorganized in Ccdc38-KO sperm. Further analyses reveal that Ccdc38 knockout causes a decreased level of TEKT3, a protein associated with acrosome biogenesis, in testes and an aberrant distribution of TEKT3 in sperm. We finally show that intracytoplasmic sperm injection overcomes Ccdc38-related infertility. Our study thus reveals a previously unknown role for CCDC38 in acrosome biogenesis and provides additional evidence for the occurrence of acrosomal hypoplasia in MMAF.

Dnali1 is required for sperm motility and male fertility in mice.

BACKGROUND: The sperm flagellum is an evolutionarily conserved specialized organelle responsible for sperm motility and male fertility. Deleterious mutations in genes involved in the sperm flagellum assembly can often cause sperm motility defects and male infertility. The murine Dnali1 gene encodes a protein that is known to interact with the cytoplasmic dynein heavy chain 1. RESULTS: A Dnali1-mutated mouse model was generated by inducing a nonsense mutation in the Dnali1 gene. The Dnali1-mutated male mice presented impaired sperm motility and were completely infertile. Although no obviously abnormal sperm morphology was observed in Dnali1-mutated male mice, the ultrastructural structure of sperm flagellum was disrupted, displaying as an asymmetrical distribution of the longitudinal columns (LCs). Notably, infertile Dnali1-mutated male mice were able to obtain offspring via ICSI. CONCLUSIONS: Our results uncover a role of DNALI1 in sperm motility and male fertility in mice, and demonstrate that ICSI overcomes Dnali1-associated male infertility, thus providing guidance for the diagnosis and genetic counseling of DNALI1-associated human infertility.

RéSUMé: CONTEXTE: Le flagelle des spermatozoïdes est un organite spécialisé conservé au cours de l'évolution, responsable de la mobilité des spermatozoïdes et de la fertilité mâle. Les mutations délétères des gènes impliqués dans l'assemblage du flagelle des spermatozoïdes peuvent souvent causer des défauts de mobilité des spermatozoïdes et une infertilité mâle. Le gène murin Dnali1 code pour une protéine flagellaire connue pour interagir avec la chaîne lourde 1 de la dynéine cytoplasmique. RéSULTATS: Un modèle murin muté au niveau de Dnali1 a été généré par induction d'une mutation non-sens dans le gène Dnali1. Les souris mâles mutées au niveau de Dnali1 présentaient une altération de la mobilité des spermatozoïdes et étaient complètement infertiles. Bien qu'aucune morphologie manifestement anormale des spermatozoïdes n'ait été observée chez les souris mâles mutées au niveau de Dnali1, l'ultrastructure du flagelle des spermatozoïdes est perturbée, se présentant avec une distribution asymétrique des colonnes longitudinales. En particulier, les souris mâles infertiles mutées au niveau de Dnali1 ont pu obtenir une progéniture au moyen de l'injection intracytoplasmique de spermatozoïdes. CONCLUSIONS: Nos résultats révèlent un rôle de DNALI1 dans la mobilité des spermatozoïdes et dans la fertilité mâle chez la souris; ils montrent que l'ICSI surmonte l'infertilité mâle associée à Dnali1, fournissant ainsi des conseils pour le diagnostic et le conseil génétique de l'infertilité masculine associée à DNALI1. MOTS-CLéS: Infertilité; Mobilité des Spermatozoïdes; Asthénozoospermie; Flagelle des Spermatozoïdes; ICSI.

Experimental and molecular support for Cfap70 as a causative gene of 'multiple morphological abnormalities of the flagella' with male infertility†.

Multiple morphological abnormalities of the flagella, a severe form of asthenozoospermia, can lead to male infertility. Recent studies have implicated an association between human CFAP70 deficiency and multiple morphological abnormalities of the flagella; however, the underlying biological mechanism and supporting experimental evidence in animal models remain unclear. To address this gap, we used CRISPR/Cas9 technology to generate Cfap70-deficient mice to investigate the relationship between Cfap70 deficiency and multiple morphological abnormalities of the flagella. Our findings show that the loss of CFAP70 leads to multiple morphological abnormalities of the flagella and spermiogenesis defects. Specifically, the lack of CFAP70 impairs sperm flagellum biogenesis and head shaping during spermiogenesis. Late-step spermatids from Cfap70-deficient mouse testis exhibited club-shaped sperm heads and abnormal disassembly of the manchette. Furthermore, we found that CFAP70 interacts with DNAI1 and DNAI2; Cfap70 deficiency also reduces the level of AKAP3 in sperm flagella, indicating that CFAP70 may participate in the flagellum assembly and transport of flagellar components. These findings provide compelling evidence implicating Cfap70 as a causative gene of multiple morphological abnormalities of the flagella and highlight the consequences of CFAP70 loss on flagellum biogenesis.

Protein Production Through Microbial Conversion of Rice Straw by Multi-Strain Fermentation.

Multi-strain mixed fermentation can provide a relatively complete lignocellulosic enzyme system compared with single-strain fermentation. This study was firstly to screen strains which have a strong ability to hydrolyse rice straw (RS) enzymatically and enrich true protein (TP). Then, the conditions in the process of SSF, including the optimum inoculum size of mixed strains, inoculation ratio, and different inoculation time of N. crassa 14-8, were optimized. The experimental results showed that the highest TP content could be obtained by using N. crassa 14-8, C. utilis, and P. chrysosporium as mixed strains, and 5 mM Mn2+ and 50 mM veratryl alcohol were used as inducers of lignin peroxidase (LiP) to improve the efficiency of enzymatic hydrolysis. When N. crassa 14-8 was inoculated 1 day later than P. chrysosporium, the total inoculum size was 10%, and the optimum ratio of N. crassa 14-8 to P. chrysosporium was 1:2, the maximum TP yield (8.89%) was obtained, with 123.37% of its increase rate. This work proposed a technique with potential application in large-scale feedstuff protein conversion.

Impact of orientation and flexibility of peptide linkers on T. maritima lipase Tm1350 displayed on Bacillus subtilis spores surface using CotB as fusion partner.

Fusion protein construction often requires peptide linkers for prolonged conformation, extended stability and enzyme activity. In this study a series of fusion between Thermotoga maritima lipase Tm1350 and Bacillus subtillis coat protein CotB, comprising of several peptide linkers, with different length, flexibility and orientations were constructed. Effects of temperature, pH and chemicals were examined, on the activity of displayed enzyme. The fusion protein with longer flexible linkers L9 [(GGGGS)4] and L7 (GGGGS-GGGGS-EAAAK-EAAAK-GGGGS-GGGGS) possess 1.29 and 1.16-fold higher activity than the original, under optimum temperature and pH respectively. Moreover, spore surface displaying Tm1350 with L3 (EAAAK-GGGGS) and L9 ((GGGGS)4) showed extended thermostably, maintaining 1.40 and 1.35-fold higher activity than the original respectively, at 80 °C after 5 h of incubation. The enzyme activity of linkers with different orientation, including L5, L6 and L7 was determined, where L7 maintained 1.05 and 1.27-fold higher activity than L5 and L6. Effect of 0.1% proteinase K, bromelain, 20% ethanol and 30% methanol was investigated. Linkers with appropriate Glycine residues (flexible) showed higher activity than Alanine residues (rigid). The activity of the displayed enzyme can be improved by maintaining orientation and flexibility of peptide linkers, to evaluate high activity and stability in industrial processes.

Influences of Various Peptide Linkers on the Thermotoga maritima MSB8 Nitrilase Displayed on the Spore Surface of Bacillus subtilis.

In the present study, fusion genes composed of Thermotoga maritima MSB8 nitrilase and Bacillus subtilis 168 outer coat protein CotG were constructed with various peptide linkers and displayed on B. subtilis DB 403 spores. The successful display of CotG-nit fusion proteins on the spore surface of B. subtilis was verified by Western blot analysis and activity measurement. It was demonstrated that the fusion with linker GGGGSEAAAKGGGGS presented the highest thermal and pH stability, which is 2.67- and 1.9-fold of the fusion without linker. In addition, fusion with flexible linker (GGGGS)3 demonstrated better thermal and pH stability than fusions with linkers GGGGS and (GGGGS)2. Fusion with rigid linker (EAAAK) demonstrated better thermal stability than fusions with linkers (EAAAK)2 and (EAAAK)3. Fusions with linker (EAAAK)2 demonstrated better pH stability than fusions with linkers (EAAAK) and (EAAAK)3. In the presence of 1 mM dithiothreitol, 1% (v/v) sodium dodecyl sulfate, and 20% (v/v) ethanol, the optimal linkers of the fusions were MGSSSN, GGGGSEAAAKGGGGS, and (GGGGS)3, respectively. In summary, our results showed that optimizing the peptide linkers with different type, length, and amino acid composition of the fusion proteins would be an efficient way to maintain the stability of fusion proteins and thus improve the nitrilase display efficiency, which could provide an effective method for rational design peptide linkers of displayed nitrilase on B. subtilis.

Cations Optimization for Protein Enrichment in Rice Straw by Mixed Cultures of Neurospora crassa 14-8 and Candida utilis Using Response Surface Methodology.

This study investigated the effect of cation optimization by mixed cultures of Neurospora crassa and Candida utilis on the true protein (TP) content. Firstly, to enhance the nutritional contents of rice straw (RS), two fermentation parameters (effect of inoculation ratio and inoculation time) were optimized. It was found that when C. utilis was inoculated 60 h later than N. crassa with the inoculation ratio of 1:5 (N. crassa to C. utilis), the maximum TP yield was obtained. In order to further optimize TP content, Plackett-Burman design (PBD) and Box-Behnken design (BBD) of response surface methodology (RSM) were adopted. The results of PBD indicated that Mn2+, Zn2+, and Cu2+ were the significant variables. The optimum values for the three cations determined by the BBD were as follows: Mn2+ 0.06 g/L, Zn2+ 0.15 g/L, and Cu2+ 0.2 g/L. After the optimization of RSM, a model was proposed to predict the optimum value 10.36% confirmed by the experimental result 9.84%. The TP content increased from 3.98 to 9.84%, with 147.24% of its increase rate. This study proposed an ecofriendly and economical way to convert RS into protein-enriched livestock feed.

Effect of Linker Length and Flexibility on the Clostridium thermocellum Esterase Displayed on Bacillus subtilis Spores.

In fusion protein design strategies, the flexibility and length of linkers are important parameters affecting the bioactivity of multifunctional proteins. A series of fusion proteins with different linkers were constructed. The effect of temperature, pH, and organic solvents was investigated on the enzymatic activity. Fusion proteins with P1(PTPTPT) and P2((PTPTPT)2) linkers remained highly active with wide temperature range. At pH 9.6, the relative activity of fusion proteins with (PTPTPT)2 and S2(EGKSSGSGSESKST) linkers was 70 and 62 % (1.75 and 1.5 times of that of non-linker ones). Fusion proteins with S3((GGGGS)4) linker retained 55 % activity after 5 h of incubation at 80 °C (1.2-fold of that of non-linker fusion proteins and 1.9-fold of GGGGS-linker fusion proteins). Finally, the relative activity of fusion proteins having different linkers was increased with 20 % dimethyl sulfoxide (DMSO) and methanol; relative activity of fusion proteins with EGKSSGSGSESKST linkers was enhanced 1.5- and 2.2-fold, respectively. These results suggest that longer flexible linker can enhance the activity and stability of displayed esterase than shorter flexible linker. Optimizing peptide linkers with length, flexibility, and amino acid composition could improve the thermostability and activity of the displayed enzyme.

Orlistat response to missense mutations in lipoprotein lipase.

The human lipoprotein lipase (LPL) is a therapeutic target for obesity, and inhibition of LPL with the approved small molecule agent orlistat has been widely used in clinic to treat obesity-related health problems such as diabetes and cardiovascular diseases. However, a variety of missense mutations in LPL protein have been observed, which may cause resistance or sensitization for orlistat, largely limiting the clinical applications of orlistat in obesity therapy. Here, we integrated molecular dynamics simulations and enzyme inhibition to investigate orlistat response to 16 disorder-associated missense mutations in LPL catalytic domain. It was found that most mutations have a modest effect on orlistat binding, and only few can exert strong impact to the binding. Three unfavorable (Trp86Arg, Ile194Thr, and Glu242Lys) and two favorable (His136Arg and Gly188Glu) mutations were identified, which can alter the binding affinity and inhibitory activity of orlistat considerably. Structural and energetic analysis revealed that these potent mutations induce orlistat resistance and sensitization by directly influencing the intermolecular interaction between LPL and orlistat or by indirectly addressing allosteric effect on LPL structure.