Outer Dense Fibers Research Articles

Novel biallelic variants in DNAH1 cause multiple morphological abnormalities of sperm flagella with favorable outcomes of fertility after ICSI in Han Chinese males.

Multiple morphological abnormalities of sperm flagella is an idiopathic asthenoteratozoospermia characterized by absent, short, coiled, angulation, and irregular-caliber flagella. Genetic variants of DNAH1 gene have been identified as a causative factor of multiple morphological abnormalities of sperm flagella and intracytoplasmic sperm injection is an available strategy for infertile males with dynein axonemal heavy chain 1 defects to conceive. To identify novel variants and candidate mutant hotspots of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans. TheDNAH1variants were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning and transmission electron microscopy, and immunostaining were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic DNAH1 variants. We identified 18 different DNAH1 variants in 11 unrelated families, including nine missense variants (p.A2564T, p.T3657R, p.G1862R, p.L2296P, p.T4041I, p.L611P, p.A913D, p.R1932Q, p.R2356W) and nine loss-of-function variants (c.2301-1G>T, p.Q1518*, p.R1702*, p.D2845Mfs*2, p.P3909Rfs*33, p.Q4040Dfs*33, p.Q4058*, p.E4060Pfs*61, p.V4071Cfs*54). A total of 66.7% (12/18) of the identified variants were novel. Morphological analysis based on Papanicolaou staining and scanning electron microscopy demonstrated the typical multiple morphological abnormalities of sperm flagella characteristics of dynein axonemal heavy chain 1-deficient spermatozoa. Immunostaining further revealed the absence of inner dynein arms but not outer dynein arms, which induced a general ultrastructural disorganization, such as the loss of central pair and mis-localization of the microtubule doublets and outer dense fibers. To date, seven affected couples have accepted the intracytoplasmic sperm injection treatment, and three of them have given birth to five healthy babies. These findings further expand the variant spectrum of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans, thus providing new information for the molecular diagnosis of asthenoteratozoospermia. The favorable fertility outcomes of intracytoplasmic sperm injection will facilitate the genetic counseling and clinical treatment of infertile males with multiple morphological abnormalities of sperm flagella in the future.

Deficiency of the Tmem232 Gene Causes Male Infertility with Morphological Abnormalities of the Sperm Flagellum in Mice.

The axoneme and accessory structures of flagella are critical for sperm motility and male fertilization. Sperm production needs precise and highly ordered gene expression to initiate and sustain the many cellular processes that result in mature spermatozoa. Here, we identified a testis enriched gene transmembrane protein 232 (Tmem232), which is essential for the structural integrity of the spermatozoa flagella axoneme. Tmem232 knockout mice were generated for in vivo analyses of its functions in spermatogenesis. Phenotypic analysis showed that deletion of Tmem232 in mice causes male-specific infertility. Transmission electron microscopy together with scanning electron microscopy were applied to analyze the spermatozoa flagella and it was observed that the lack of TMEM232 caused failure of the cytoplasm removal and the absence of the 7th outer microtubule doublet with its corresponding outer dense fiber (ODF). Co-IP assays further identified that TMEM232 interacts with ODF family protein ODF1, which is essential to maintain sperm motility. In conclusion, our findings indicate that TMEM232 is a critical protein for male fertility and sperm motility by regulating sperm cytoplasm removal and maintaining axoneme integrity.

Structural specializations of the sperm tail

Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm are powered by a microtubule-based molecular machine-the axoneme-but it is unclear how axonemal microtubules are ornamented to support motility in diverse fertilization environments. Here, we present high-resolution structures of native axonemal doublet microtubules (DMTs) from sea urchin and bovine sperm, representing external and internal fertilizers. We identify >60 proteins decorating sperm DMTs; at least 15 are sperm associated and 16 are linked to infertility. By comparing DMTs across species and cell types, we define core microtubule inner proteins (MIPs) and analyze evolution of the tektin bundle. We identify conserved axonemal microtubule-associated proteins (MAPs) with unique tubulin-binding modes. Additionally, we identify a testis-specific serine/threonine kinase that links DMTs to outer dense fibers in mammalian sperm. Our study provides structural foundations for understanding sperm evolution, motility, and dysfunction at a molecular level.

BBOF1 is required for sperm motility and male fertility by stabilizing the flagellar axoneme in mice.

The sperm flagellum is a specialized type of motile cilium composed of a typical "9 + 2" axonemal structure with peri-axonemal structures, such as outer dense fibers (ODFs). This flagellar arrangement is crucial for sperm movement and fertilization. However, the association of axonemal integrity with ODFs remains poorly understood. Here, we demonstrate that mouse BBOF1 could interact with both MNS1, an axonemal component, and ODF2, an ODF protein, and is required for sperm flagellar axoneme maintenance and male fertility. BBOF1 is expressed exclusively in male germ cells from the pachytene stage onwards and is detected in sperm axoneme fraction. Spermatozoa derived from Bbof1-knockout mice exhibit a normal morphology, however, reduced motility due to the absence of certain microtubule doublets, resulting in the failure to fertilize mature oocytes. Furthermore, BBOF1 is found to interact with ODF2 and MNS1 and is also required for their stability. Our findings in mice suggest that Bbof1 could also be essential for human sperm motility and male fertility, thus is a novel potential candidate gene for asthenozoospermia diagnosis.

STK33 Phosphorylates Fibrous Sheath Protein AKAP3/4 to Regulate Sperm Flagella Assembly in Spermiogenesis

Spermatogenesis defects are important for male infertility; however, the etiology and pathogenesis are still unknown. Herein, we identified two loss-of-function mutations of STK33 in seven individuals with non-obstructive azoospermia. Further functional studies of these frameshift and nonsense mutations revealed that Stk33-/KI male mice were sterile, and Stk33-/KI sperm were abnormal with defects in the mitochondrial sheath, fibrous sheath, outer dense fiber, and axoneme. Stk33KI/KI male mice were subfertile and had oligoasthenozoospermia. Differential phosphoproteomic analysis and invitro kinase assay identified novel phosphorylation substrates of STK33, fibrous sheath components A-kinase anchoring protein 3 and A-kinase anchoring protein 4, whose expression levels decreased in testis after deletion of Stk33. STK33 regulated the phosphorylation of A-kinase anchoring protein 3/4, affected the assembly of fibrous sheath in the sperm, and played an essential role in spermiogenesis and male infertility.

Biallelic variants in IQCN cause sperm flagellar assembly defects and male infertility.

What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? Deficiency in IQCN causes sperm flagellar assembly defects and male infertility. The manchette is a transient structure that is involved in the shaping of the human spermatid nucleus and protein transport within flagella. Our group recently reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN lead to total fertilization failure and defective acrosome structure phenotypes. However, the function of IQCN in sperm flagellar assembly is still unknown. Fifty men with infertility were recruited from a university-affiliated center from January 2014 to October 2022. Genomic DNA was extracted from the peripheral blood samples of all 50 individuals for whole-exome sequencing. The ultrastructure of the spermatozoa was assessed by transmission electron microscopy. Computer-assisted sperm analysis (CASA) was used to test the parameters of curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). An Iqcn knockout (Iqcn-/-) mouse model was generated by CRISPR-Cas9 technology to evaluate sperm motility and the ultrastructure of the flagellum. Hyperactivation and sperm fertilizing ability were assessed in a mouse model. Immunoprecipitation followed by liquid chromatography-mass spectrometry was used to detect IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our cohort of infertile men. The sperm from the affected individuals showed an irregular '9 + 2' structure of the flagellum, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn-/- male mice. VSL, VCL, and VAP in the sperm of Iqcn-/- male mice were significantly lower than those in Iqcn+/+ male mice. Partial peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or a chaotic arrangement of DMTs was observed in the principal piece and end piece of the sperm flagellum. Hyperactivation and IVF ability were impaired in Iqcn-/- male mice. In addition, we investigated the causes of motility defects and identified IQCN-binding proteins including CDC42 and the intraflagellar transport protein families that regulate flagellar assembly during spermiogenesis. More cases are needed to demonstrate the relation between IQCN variants and phenotypes. Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, providing a genetic marker for sperm motility deficiency and male infertility. This work was supported by the National Natural Science Foundation of China (81974230 and 82202053), the Changsha Municipal Natural Science Foundation (kq2202072), the Hunan Provincial Natural Science Foundation (2022JJ40658), and the Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya (YNXM-202114 and YNXM-202201). No conflicts of interest were declared. N/A.

Characterization of a spontaneously occurring self-reactive antibody against sperm in mice

We previously established a spontaneously occurring monoclonal antibody, namely Ts3, that was reactive to sperm from an aged male mouse. The present study investigated the characteristic properties and reproductive functions of Ts3. Immunofluorescent staining revealed that Ts3 reacted to epididymal sperm, and the corresponding antigen was located in the midpiece and principal piece. Immunohistochemistry revealed positive reactions in the germ cells and Sertoli cells in the testis, the epithelial cells in the epididymis and vas deferens. Through western blotting with two-dimensional electrophoresis, we demonstrated that Ts3 reacted with four spots, which were around Mr ∼25,000–60,000 and pI 5–6. MALDI-TOF/TOF mass spectrometry identified outer dense fiber 2 (ODF2) as a candidate for Ts3. ODF2 is a cytoskeletal structural component located in the midpiece and principal piece of the flagella of mammalian sperm. This was validated with the result of immunofluorescent staining, suggesting that ODF2 was the main target antigen for Ts3. Sperm immobilization test showed that Ts3 possessed sperm immobilizing activity. Furthermore, Ts3 impaired early embryo development but not in vitro fertilization. These results suggest that ODF2 plays an important role in both sperm function and early embryonic development.

The association of ODF4 with AK1 and AK2 in mice is essential for fertility through its contribution to flagellar shape

Normal sperm flagellar shape and movement are essential for fertilization. The integral protein outer dense fiber 4 (ODF4) localizes to ODFs, but its function remains unclear. Adenylate kinase (AK) is a phosphotransferase that catalyzes the interconversion and controls the concentration equilibrium of adenine nucleotides. AK shuttles ATP to energy-consuming sites. Here, we report on the relationship of flagellar shape and movement with ODF4, AK1 and AK2 by using Odf4-deletion (Odf4−/−) mice. Soluble ODF4 is coimmunoprecipitated with AK1 and AK2 in Odf4+/+ spermatozoa. ODF4, AK1 and AK2 localize to whole flagella (plasmalemma, mitochondria, ODFs, and residual cytoplasmic droplets (CDs)), principal pieces, and midpieces, respectively. Odf4−/− sperm flagella lose ODF4 and reduce AK1 and AK2 but produce ATP. The flagellum is bent (hairpin flagellum) with a large CD in the midpiece. There is no motility in the midpiece, but the principal piece is motile. Odf4−/− spermatozoa progress backward and fail to ascend in the uterus. Thus, Odf4−/− males are infertile owing to abnormal flagellar shape and movement caused mainly by the loss of ODF4 with AK1 and AK2. This study is supported by the rescue experiment; the abnormalities and male infertility caused by Odf4 deletion were reversed by Odf4 restoration.

ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models.

WEE1 has emerged as an attractive target in epithelial ovarian cancer (EOC), but how EOC cells may alter their sensitivity to WEE1 inhibition remains unclear. Here, through a cell cycle machinery-related gene RNAi screen, we found that targeting outer dense fiber of sperm tails 2-like (ODF2L) was a synthetic lethal partner with WEE1 kinase inhibition in EOC cells. Knockdown of ODF2L robustly sensitized cells to treatment with the WEE1 inhibitor AZD1775 in EOC cell lines in vitro as well as in xenografts in vivo. Mechanistically, the increased sensitivity to WEE1 inhibition upon ODF2L loss was accompanied by accumulated DNA damage. ODF2L licensed the recruitment of PKMYT1, a functionally redundant kinase of WEE1, to the CDK1-cyclin B complex and thus restricted the activity of CDK1 when WEE1 was inhibited. Clinically, upregulation of ODF2L correlated with CDK1 activity, DNA damage levels, and sensitivity to WEE1 inhibition in patient-derived EOC cells. Moreover, ODF2L levels predicted the response to WEE1 inhibition in an EOC patient-derived xenograft model. Combination treatment with tumor-targeted lipid nanoparticles that packaged ODF2L siRNA and AZD1775 led to the synergistic attenuation of tumor growth in the ID8 ovarian cancer syngeneic mouse model. These data suggest that WEE1 inhibition is a promising precision therapeutic strategy for EOC cells expressing low levels of ODF2L.

Decoding the role of cytochrome c in metabolism of human spermatozoa by Raman imaging.

The normal functioning of sperm cells requires cytochrome c in the redox balanced forms: reduced and oxidized. The oxidized form of cytochrome c is localized in the mitochondrial intermembrane space and is a part of the electron transport chain. This ensures that electron shuttling between the complex III, cytochrome c, and complex IV can occur leading to controlled effective oxidative phosphorylation (respiration) and ATP production needed for most steps in spermatozoal maturation, motility, hyperactivation and fertilization. We studied the biochemical composition of specific organelles in sperm cells by Raman imaging. The structures of the head consisting of the nucleus and acrosome, the midpiece representing mitochondria, and the tail characterized by the sperm axoneme surrounded by outer dense fiber and covered by the membrane were measured. Metabolic biochemical analysis of mitochondria, head and tail of sperm cells, and seminal plasma by using Raman imaging combined with chemometric classification method of Cluster Analysis has been obtained. Our results show that cytochrome c, which is a key protein that is needed to maintain life (respiration) and cell death (apoptosis), is located in sperm mitochondria in the oxidized or reduced form of the heme group. This work demonstrated that an application of Raman micro-spectroscopy can be extended to monitoring the redox state of mitochondrial cytochrome c in sperm cells.

IQUB deficiency causes male infertility by affecting the activity of p-ERK1/2/RSPH3.

Can new genetic factors responsible for male infertility be identified, especially for those characterized by asthenospermia despite normal sperm morphology? We identified the novel pathogenetic gene IQ motif and ubiquitin-like domain-containing (IQUB) as responsible for male infertility characterized by asthenospermia, involving sperm radial spoke defects. To date, only a few genes have been found to be responsible for asthenospermia with normal sperm morphology. Iqub, encoding the IQUB protein, is highly and specifically expressed in murine testes and interacts with the proteins radial spoke head 3 (RSPH3), CEP295 N-terminal like (CEP295NL or DDC8), glutathione S-transferase mu 1 (GSTM1) and outer dense fiber of sperm tails 1 (ODF1) in the yeast two-hybrid system. The IQUB variant was identified by whole-exome sequencing in a cohort of 126 male infertility patients with typical asthenospermia recruited between 2015 and 2020. Knockout (KO) and knockin (KI) mouse models, scanning and transmission electron microscopy (TEM), and other functional assays were performed, between 2019 and 2021. The IQUB variant was identified by whole-exome sequencing and confirmed by Sanger sequencing. Iqub KO and KI mice were constructed to mimic the phenotype of the affected individual. After recapitulating the phenotype of human male infertility, scanning and TEM were performed to check the ultrastructure of the sperm. Western blot and co-immunoprecipitation were performed to clarify the pathological mechanism of the IQUB variant. We identified a homozygous nonsense IQUB variant (NM_001282855.2:c.942T> G(p.Tyr314*)) from an infertile male. Iqub KO and KI mice mimicked the infertility phenotype and confirmed IQUB to be the pathogenetic gene. Scanning and TEM showed that sperm of both the mouse models and the affected individual had radial spoke defects. The functional assay suggested that IQUB may recruit calmodulin in lower Ca2+ environments to facilitate the normal assembly of radial spokes by inhibiting the activity of RSPH3/p-ERK1/2 (a nontypical AKAP (A-Kinase Anchoring Protein) forming by RSPH3 and phosphorylation of extracellular signal-regulated kinase 1 and 2 (p-ERK1/2)). Additional cases are needed to confirm the genetic contribution of IQUB variants to male infertility. In addition, because no IQUB antibody is available for immunofluorescence and the polyclonal antibody we generated was only effective in western blotting, immunostaining for IQUB was not performed in this study. Therefore, this study lacks direct in vivo proof to confirm the effect of the variant on IQUB protein level. Our results suggest a causal relation between IQUB variants and male infertility owing to asthenospermia, and partly clarify the pathological mechanism of IQUB variants. This expands our knowledge of the genes involved in human sperm asthenospermia and potentially provides a new genetic marker for male infertility. This work was supported by the National Key Research and Development Program of China (2021YFC2700100), the National Natural Science Foundation of China (32130029, 82171643, 81971450, 82001538, and 81971382) and the Guangdong Science and Technology Department Guangdong-Hong Kong-Macao Joint Innovation Project (2020A0505140003). There are no competing interests to declare. N/A.

Bull sperm and seminal plasma proteins and their relationship with fertility: a review

The efficiency of artificial insemination (AI) is greatly influenced by the quality of semen. Spermatozoa and seminal plasma are found in semen, which play a role in the reproductive process and its ability to fertilize an egg and maintain the development of an embryo. Various factors will determine the fertility capacity of a sperm, both from the intrinsic factors of the sperm and the plasma component of the semen. Seminal plasma proteins are crucial for maintaining the stability of the membrane, viability, motility of spermatozoa, acrosome reactions, maintaining osmotic pressure and helping the fertilization process. Good quality semen will support the fertilization process. The purpose of this scoping review is to increase our understanding of protein from sperm and seminal plasma of bulls and their relationship with fertility. The sperm proteins that were significantly correlated with fertility were Outer Dense Fiber protein 2 (ODF2), Protamine (PRM), Testis specific histine 2B (TH2B), Phosphatidylethanolamine binding protein (PEBP4), and Ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRSC2). Meanwhile, the seminal plasma proteins positively correlated with fertility were Osteopontin (OPN), Phospholipasea 2 (PLA2), P25b, Acidic seminal fluid proteins (aSFP), Alpha-L-fucosidase (a-L-fucosidase), and Binder of sperm (BSPs).

CAMP and the Fibrous Sheath Protein CABYR (Ca2+-Binding Tyrosine-Phosphorylation-Regulated Protein) Is Required for 4D Sperm Movement

A new life starts with successful fertilization whereby one sperm from a pool of millions fertilizes the oocyte. Sperm motility is one key factor for this selection process, which depends on a coordinated flagellar movement. The flagellar beat cycle is regulated by Ca2+ entry via CatSper, cAMP, Mg2+, ADP and ATP. This study characterizes the effects of these parameters for 4D sperm motility, especially for flagellar movement and the conserved clockwise (CW) path chirality of murine sperm. Therefore, we use detergent-extracted mouse sperm and digital holographic microscopy (DHM) to show that a balanced ratio of ATP to Mg2+ in addition with 18 µM cAMP and 1 mM ADP is necessary for controlled flagellar movement, induction of rolling along the long axis and CW path chirality. Rolling along the sperm’s long axis, a proposed mechanism for sperm selection, is absent in sea urchin sperm, lacking flagellar fibrous sheath (FS) and outer-dense fibers (ODFs). In sperm lacking CABYR, a Ca2+-binding tyrosine-phosphorylation regulated protein located in the FS, the swim path chirality is preserved. We conclude that specific concentrations of ATP, ADP, cAMP and Mg2+ as well as a functional CABYR play an important role for sperm motility especially for path chirality.

Effect of ozone versus naringin on testicular injury in experimentally induced ulcerative colitis in adult male albino rats

ABSTRACT Testicular dysfunction is caused by the continuous inflammation and oxidative stress that are present at the local site in ulcerative colitis (UC) spreading to the testes via systemic circulation. The influence of ozone and naringine on colitis-mediated testicular dysfunction was investigated in this study. Forty-eight adult male rats were divided into four groups: I control group, II dextran sodium sulfate (DSS) UC-induced group, III DSS+naringine, and IV DSS+ozone groups. UC was induced in groups II, III, and IV using 0.1 ml of 4% DSS in their drinking water per day for 6 days by gastric gavage. All animals were sacrificed 45 days from the start. Blood samples were obtained to estimate serum testosterone hormone. Testicular tissues were processed for measurement of tissue malondialdehyde (MDA) and examined by light and electron microscopes. Ultrastructurally, group II revealed a relatively thick basement membrane enveloping the seminiferous tubule. Sertoli cell cytoplasm appears rarified with wide intracellular spaces, vacuoles, and multiple lysosomes; distorted spermatogonia with electron dense nuclei and cytoplasm; and primary spermatocytes with small nuclei and electron dense cytoplasm. Abnormal sperm profiles were visible in middle pieces, mid, principle, and end pieces that were markedly affected with disorganization of axoneme and outer dense fibers. Leydig cells revealed dilated cisternae of smooth endoplasmic reticulum. Morphometric and statistical analyses were performed. Group III showed some improvement; however, group IV showed more improvement. The results indicated that ozone caused marked improvement than naringine against UC-induced testicular damage via their antioxidant and anti-inflammatory properties.

OP-17 - Characterization of spontaneously-occurring self-reactive antibody to sperm and its effects on in-vitro fertilization

Anti-sperm antibodies detected in humans possibly cause infertility. The mechanism of antibody production and the corresponding antigen are still unknown. Previously, we established a monoclonal antibody (named Ts3) reactive to sperm from an aged male mouse without any immunization. This study aims to identify the target self-antigen of Ts3 and to examine its effects on in-vitro fertilization. To examine the localization of the Ts3 recognizing antigen, mouse epididymal sperm were collected for immunofluorescent staining. The sperm proteins were extracted for two-dimensional electrophoresis and western blot analysis. Positive spots were subjected to mass spectrometry (MS). To determine influence on sperm motility, sperm immobilization test (SIT) with Ts3 was carried out. Further, the effects of Ts3 on mouse in-vitro fertilization and the developmental capacity of preimplantation embryos were investigated. Ts3 reacted to the midpiece and principal piece of mouse sperm. The detected molecular weight of the antigen to Ts3 was around 37 kDa, and the molecule was identified as outer dense fiber protein 2 (ODF2) by MS. In SIT, Ts3 was suggested to have harmful effect on sperm motility. Ts3 inhibited embryo development but not fertilization in mouse. Immunofluorescent staining of mouse preimplantation embryo showed Ts3 reacted to the membrane of the embryo and the areas of cellular contact. This study showed that Ts3, a spontaneously-occurring self-reactive antibody to sperm, recognized ODF2 as a targeted antigen. Although Ts3 did not inhibit fertilization, it impaired the sperm motility and the development of preimplantation embryo. Therefore, ODF2 antigen may be a pathogenic factor in infertility.

Alginate oligosaccharides increase boar semen quality by affecting gut microbiota and metabolites in blood and sperm.

Alginate oligosaccharides (AOS), natural polymers from brown seaweeds (such as Laminaria japonica, Undaria pinnatifida, and Sargassum fusiforme), have been reported to possess many beneficial advantages for health. In the current study, after 9 weeks of dietary supplementation, AOS 10 mg/kg group (AOS 10) group increased boar sperm motility from 87.8% to 93.5%, p < 0.05. Moreover, AOS10 increased the relative abundances of Bifidobacterium, Coprococcus, Butyricicoccus (1.3–2.3-fold; p < 0.05) to increase the beneficial blood and sperm metabolites (1.2–1.6-fold; p < 0.05), and important sperm proteins such as gelsolin, Zn-alpha2 glycoprotein, Cation Channel Sperm-Associated Protein, outer dense fiber of sperm tails, etc. (1.5–2.2-fold; p < 0.05). AOS had a long-term beneficial advantage on boar semen quality by the increase in semen volume (175 vs. 160 ml/ejaculation, p < 0.05). AOS may be used as dietary additives for improving semen quality.

Novel mutations of PMFBP1 in a man with acephalic spermatozoa defects.

BackgroundAcephalic spermatozoa (AS) is a serious but rare reproductive genetic disorder that causes infertility in men. To date, only a few genes associated with AS defects have been identified, including the polyamine modulated factor 1 binding protein 1 (PMFBP1) gene. Consistent with this, PMFBP1 localizes to the head–neck connection, which bridges the implantation fossa and basal body.MethodsA male patient was diagnosed as having an AS defect. Blood samples from all family members and a sample of the patient's semen were collected to determine the genetic causes of his infertility.ResultsCompound heterozygote mutation in the PMFBP1 gene, which is associated with AS defects in the present case: two loss‐of‐function mutations, with one a nonsense mutation c.361C > T p.Gln121Ter, and another a splice donor mutation c.414 + 1G > T. The current study, together with previous studies, suggests that the nonsense mutation is responsible for a truncated PMFBP1 protein during its formation; a splice donor mutation c.414 + 1G > T might lead to new open reading frames, from which the dysfunction of an abnormal PMFBP1 protein might be predicted. Additionally, the expression of outer dense fiber 1 (ODF1) and ODF2 proteins has been experimentally shown to be regulated by the truncated PMFBP1 protein.ConclusionWe herein present a case with AS defects associated with heterozygote mutations of PMFBP1, which have been shown to be rare and pathogenic; the association with an AS defect is a monogenic disorder with a recessive inherited pattern in the patient's family.

Gamma-oryzanol supplemented in extender enhances the quality of semen cryopreservation and alters proteomic profile in Thai swamp buffalo

Reactive oxygen species (ROS) exert an adverse effect on sperm quality during the freezing process. Gamma-oryzanol is an effective antioxidant and has the ability to inhibit lipoperoxidation in various cells. Therefore, this study aims to investigate the effect of gamma-oryzanol supplementation in extender on post-thawed motility and proteomic profiles of swamp buffalo spermatozoa. Each ejaculate of an individual bull was divided into four equal aliquots. Gamma-oryzanol was supplemented at 0 (control), 0.1, 0.25, and 0.5 mM in tris-citrate egg yolk extender. The parameters of sperm motility were evaluated using computer assisted semen analyzer (CASA). The results showed that the progressive motility was significantly higher in 0.5 mM of gamma-oryzanol supplementation group when compared with the control group (p < 0.05), but no significant differences were observed among the treatments. In addition, a proteomic approach was applied to analyze the differentially expressed proteins in post-thawed sperm with or without gamma-oryzanol supplementation in extender. We confirmed that 2-phospho-d-glycerate hydro-lyase (ENO1), glutathione s-transferase mu 1 (GSTM1), phospholipid hydroperoxide glutathione peroxidase (GPX4), outer dense fiber protein 2 (ODF2), tektin-4 (TEKT4), tubulin beta-4B chain (TUBB4B), and ATP synthase subunit beta (ATP5B) were up-regulated in 0.5 mM of gamma-oryzanol supplementation group, which might be associated with the improved post-thawed motility observed in this treatment group. These results demonstrate the beneficial effect of gamma-oryzanol on post-thawed survival of swamp buffalo spermatozoa and help advance the understanding about molecular metabolism of sperm in this species.

Changes in the testicular histomorphometry and their association with genes expression pattern of testes from birth to puberty in Beetal goat kids

In the present study, twenty-eight (n = 28) longitudinally rear male goat kids were slaughtered at monthly intervals (n = 4/group) till the 6th month of age; to examine the testicular development and establishment of spermatogenesis through histo-architecture along with mRNA expression of related genes. The results showed that seminiferous tubular diameter was linearly increased (P < 0.001) at birth (40 µm) to 6th months (200 µm). The epithelial height was 18.41 ± 0.65 µm at birth and 164 ± 2.82 µm at 6th months. Whereas, at 3rd months age greatest increase was observed (74.08 ± 1.66 µm), possibly due to a rapid increase in the numbers of spermatocytes and spermatids (4.0 ± 0.3 and 0.9 ± 0.04, respectively). At the same time point (4–6 months of age), a dramatic increase (P < 0.001) was observed in the mRNA level of genes expressed in haploid germ cells including Protamine (PRM1), Outer dense fiber protein (ODF2) and Stimulated by Retinoic Acid (STRA8). In addition, the maturation of Sertoli cells (SCs) was appeared to be completed at the start of 1st month (5.6 ± 0.5), which was further validated by sudden repression (P < 0.001) of SC-specific SOX9 and FSHR mRNA levels during this period. The spermatids that appeared at the end of the 3rd month reflect the start of spermatogenesis. However, a significant (P < 0.001) number of elongated spermatids and mature spermatozoa was observed at the end of 6 months, which indicated pubertal onset (9.8 ± 0.7 and 9.9 ± 0.9, respectively). The mRNA expression of other genes altered during this developmental phase was Heat-shock Protein 8 (HSPA8) and Aromatase (CYP19A1), both was important in sperm maturation and testosterone level. In conclusion, marked changes in cellular and molecular events were observed from the 3rd month; when active spermatogenesis initiated, and mature spermatozoa appeared in the 6th month at pubertal onset in Beetal bucks.

Homozygous variants in AKAP3 induce asthenoteratozoospermia and male infertility

BackgroundAs a common type of asthenoteratozoospermia, multiple morphological abnormalities of the sperm flagella (MMAF) can cause male infertility. Previous studies have revealed genetic factors as a major cause of MMAF....