Sperm Structure Research Articles

Morphology and morphometry of sperm in Kurdish stallions, a local breed from western Iran.

The present work was designed for a thorough investigation into the sperm morphology and morphometry of Kurdish stallions. The semen samples were collected from 10 Kurdish stallions. Three preparations from each ejaculate were stained with eosin-nigrosin (EN), Diff-Quik (DQ) and Rose Bengal (RB). The area, perimeter, length and width of the sperm head as well as tail length and total sperm length were measured. The parameters ellipticity, elongation, roughness and regularity were calculated. The morphology of sperm was also investigated under scanning and transmission electron microscopes. DQ and RB provided more clarified images for examining sperm structures compared to the EN method. The head length, head width, area and perimeter in EN were significantly higher than those in DQ and RB (p ≤ .05). Furthermore, the difference in head width, head area and head perimeter between DQ and RB was not significant (p ≥ .05). The tail length and total sperm length in all methods were close together (p ≥ .05). The highest percentage of normal sperm was seen in DQ and RB methods (82.55 ± 2.88 and 88.31 ± 5.19) respectively. The highest values for ellipticity, elongation and regularity were found in RB, whereas the highest value for roughness was measured in EN. Tail defects including coiled tails, and folded midpieces were the most frequent. Scanning electron microscope revealed two types of head shapes: heads with round anterior border, and heads with flat anterior border. The results indicated that despite the routine use of EN for morphological assessment of stallion sperm, RB and DQ can be considered for more clarified details of sperm structure including acrosome and midpiece. Furthermore, the Kurdish stallion sperm has morphometric traits in the normal range established for stallions; yet, some traits were larger than those reported for other breeds. It seems that the sperm of the Kurdish stallion has a longer head and tail in comparison with other horse breeds.

Sambucus Nigra: black fruits that makes sperms come out ahead in in vitro fertilization

Freezing is a non-physiological method that includes a series of processes such as dilution, incubation, cooling, freezing and thawing, which affect the structure, biochemistry and function of sperm. Human sperm freezing has overcome many spatial and temporal limitations and is now an integral part of assisted reproductive technologies (ART). Freezing causes biochemical, functional and structural changes in sperm. These changes include plasma and sperm acrosome membranes, which increase their permeability and leads to disruption of morphology, motility and chromatin structure. Therefore, freezing causes irreversible damage to sperm, which leads to the loss of movement and biological function of most sperm. Sperm health is one of the important factors in the fertility of couples. Therefore, reducing the harmful effects caused by freezing and properly performing the in vitro Fertilization (IVF) process, the development of useful solutions is inevitable. The extract from black fruits of Elderberry (Sambucus nigra) has potent in vitro effect on the sperm motility, vitality and oxidative profile. This extract can largely improve the sperms parameters and stereological parameters (spermatogonia, primary spermatocyte, round spermatid, and Leydig cells), decrease of TNF-a and caspase-3 expression, increase of the serum testosterone, increase of mitochondrial activity, and reduction of reactive oxygen species. So, it can also be used as in vivo status. We suggested that black fruits of Sambucus nigra makes sperms come out ahead in IVF techniques.

The sperm structure of Clinidium canaliculatum (Costa): A contribution to the systematic position of Rhysodidae (Coleoptera: Carabidae)

The systematic position and the phylogenetic relationship of Rhysodidae members is still debated, with some authors considering the group as a separate family of Adephaga, while for others they could be a subfamily of Carabidae. The group have morphological traits quite different from Carabidae and an aberrant behaviour compared to ground beetles being not predaceous. The sperm ultrastructure of C. canaliculatum was studied comparatively with other species of beetles, Carabidae in particular. The results indicate that the sperm structure of this species is similar to that of the Carabinae species. As in these species, C. canaliculatum has sperm conjugates with an apical conical cap protecting the heads and the initial region of flagella. This sperm appearance is also shared by another species of Rhysodidae, Omoglymmius hamatus. The material of the apical cap consists of an electron-dense material with a peculiar outer net configuration. Many species of Carabidae, however, can present a different type of sperm conjugation, the spermatostyle: a long rod-like structure where the individual sperms have only the most apical part inserted in the cortical area and the flagella are completely free. C. canaliculatum sperm are endowed with a mono-layered acrosome, a nucleus of variable shape along its length, a flagellum consisting of a typical axoneme 9 + 9+2, provided with 16 protofilaments in the tubular wall of accessory tubules, two asymmetric mitochondrial derivatives with the left one larger than the opposite one, and the right accessory body elongated and larger than the opposite one. These sperm characteristics, which are shared also by another member of the group, suggest the demotion of the family Rhysodidae to the subfamily Rhysodinae within Carabidae, a result also supported by recent molecular data.

The protective effect of zinc oxide and selenium oxide nanoparticles on the functional parameters of rat sperm during vitrification.

While sperm freezing (cryopreservation) is an effective method for preserving fertility, it can potentially harm the structure and function of sperm due to an increase in the production of reactive oxygen species. This study aimed to assess the impact of zinc oxide nanoparticles (ZnONPs) and selenium oxide nanoparticles (SeONPs) on various sperm functional parameters, including motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), acrosome membrane integrity (ACi), and malondialdehyde (MDA) levels. Semen samples were collected from 20 Albino Wistar rats. These samples were then divided into six groups: fresh, cryopreservation control, and groups supplemented with SeONPs (1, 2, 5 μg/mL) and ZnONPs (0.1, 1, 10 μg/mL). Statistical analysis revealed that all concentrations of SeONPs increased total motility and progressive reduction of MDA levels compared to the cryopreservation control group (p<0.05). However, supplementation with ZnONPs did not affect these parameters (p>0.05). Conversely, supplements of 1 and 2 μg/mL SeONPs and 1 μg/mL ZnONPs contributed to the improvement of PMI and ACi (p<0.05). Yet, no significant change was observed in MMP with any concentration of SeONPs and ZnONPs compared to the cryopreservation control group (p>0.05). The findings suggest that optimal concentrations of SeONPs may enhance sperm parameters during the freezing process.

Deleterious genetic changes in AGTPBP1 result in teratozoospermia with sperm head and flagella defects.

Approximately 10%-15% of couples worldwide are infertile, and male factors account for approximately half of these cases. Teratozoospermia is a major cause of male infertility. Although various mutations have been identified in teratozoospermia, these can vary among ethnic groups. In this study, we performed whole-exome sequencing to identify genetic changes potentially causative of teratozoospermia. Out of seven genes identified, one, ATP/GTP Binding Protein 1 (AGTPBP1), was characterized, and three missense changes were identified in two patients (Affected A: p.Glu423Asp and p.Pro631Leu; Affected B: p.Arg811His). In those two cases, severe sperm head and tail defects were observed. Moreover, AGTPBP1 localization showed a fragmented pattern compared to control participants, with specific localization in the neck and annulus regions. Using murine models, we found that AGTPBP1 is localized in the manchette structure, which is essential for sperm structure formation. Additionally, in Agtpbp1-null mice, we observed sperm head and tail defects similar to those in sperm from AGTPBP1-mutated cases, along with abnormal polyglutamylation tubulin and decreasing △-2 tubulin levels. In this study, we established a link between genetic changes in AGTPBP1 and human teratozoospermia for the first time and identified the role of AGTPBP1 in deglutamination, which is crucial for sperm formation.

Sperm models in European Plecoptera

Systematic issues regarding Plecoptera are still debated, and the molecular data seem to be unable to definitively clarify the relationships within the order. Spermatozoa are under constant evolutionary pressure, and comparative spermatology can be useful in carrying systematic and phylogenetic information. In the present paper we describe the sperm structure, using light, scanning and transmission electron and immunofluorescence microscopy, of six Euholognatha species belonging to genera not analyzed in our previous studies, i.e. Capnopsis, Amphinemura, Rhabdiopteryx, Tyrrhenoleuctra, Zwicknia and Protonemura. The spermatozoa of all the species examined are fîliform and have a flagellum characterized by an axoneme with 9 + 9+2 pattern and two mitochondrial derivatives. Their ultrastructure shows a degree of heterogeneity within the order. On the contrary, morphological features of sperm are well conserved inside a single Euholognathan family, and the species share a general family sperm model, even if different interspecific or intergeneric characters can be identified and used for systematic inferences. Among Nemouroidea, Taeniopterygidae, showing a peculiar sperm model, seems to have an isolated phylogenetic position. Nemouridae, with a mono-layered acrosome, are isolated among the remaining families, while we can hypothesize a sister taxa relationship between Leuctridae and Capniidae. As regards Perloidea, the sperm characters suggest a closer relationship between Chloroperlidae and Perlodidae, rather than between Perlidae and Perlodidae, as commonly hypothesized.

Deficiency in AK9 causes asthenozoospermia and male infertility by destabilising sperm nucleotide homeostasis

Asthenozoospermia is the primary cause of male infertility; however, its genetic aetiology remains poorly understood. Adenylate kinase 9 (AK9) is highly expressed in the testes of humans and mice and encodes a type of adenosine kinase that is functionally involved in cellular nucleotide homeostasis and energy metabolism. We aimed to assess whether AK9 is involved in asthenozoospermia. One-hundred-and-sixty-five Chinese men with idiopathic asthenozoospermia were recruited. Whole-exome sequencing (WES) and Sanger sequencing were performed for genetic analyses. Papanicolaou staining, Haematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy were used to observe the sperm morphology and structure. Ak9-knockout mice were generated using CRISPR-Cas9. Sperm adenosine was detected by liquid chromatography-mass spectrometry. Targeted sperm metabolomics was performed. Intracytoplasmic sperm injection (ICSI) was used to treat patients. We identified five patients harbouring bi-allelic AK9 mutations. Spermatozoa from men harbouring bi-allelic AK9 mutations have a decreased ability to sustain nucleotide homeostasis. Moreover, bi-allelic AK9 mutations inhibit glycolysis in sperm. Ak9-knockout male mice also presented similar phenotypes of asthenozoospermia. Interestingly, ICSI was effective in bi-allelic AK9 mutant patients in achieving good pregnancy outcomes. Defects in AK9 induce asthenozoospermia with defects in nucleotide homeostasis and energy metabolism. This sterile phenotype could be rescued by ICSI. The National Natural Science Foundation of China (82071697), Medical Innovation Project of Fujian Province (2020-CXB-051), open project of the NHC Key Laboratory of Male Reproduction and Genetics in Guangzhou (KF202004), Medical Research Foundation of Guangdong Province (A2021269), Guangdong Provincial Reproductive Science Institute Innovation Team grants (C-03), and Outstanding Young Talents Program of Capital Medical University (B2205).

Zinc oxide and selenium nanoparticles can improve semen quality and heat shock protein expression in cryopreserved goat (Capra hircus) spermatozoa

BackgroundReactive oxygen species (ROS) are strongly linked with oxidative stress (OS) generated during the process of sperm cryopreservation. Indeed, cellular damage from ROS has been implicated during sperm cryopreservation which causes deterioration in sperm quality and antioxidant nanoparticles (NPs) have been successful in preventing such damage. The interaction of NPs with sperm cells has been less frequently explored in farm animals. ObjectiveThe present study explored the effect of NP supplementation on sperm ultrastructure, potential interaction with sperm membrane (plasma and acrosome membrane), heat shock protein (HSP) gene expression levels and sperm quality in cryopreserved buck semen. Materials and methodsThirty-two (32) ejaculates were collected from four (4) adult male bucks and then diluted in Tris- citric acid- fructose- egg yolk (TCFY) extender containing the Zinc-oxide (ZnO) and Selenium (Se) NP treatments (T0: Control; TZn: 0.1 mg/mL ZnO NPs and TSe: 1 µg/mL Se NPs) after initial evaluation. Diluted semen was packed in 0.25 mL French mini straws and then stored in liquid nitrogen (LN2). Sperm parameters, lipid peroxidation (LPO) profile, sperm head morphology ultrastructural classification under transmission electron microscope (TEM), potential interaction of NPs with sperm membrane and expression of HSP genes were evaluated in the different treatment groups. ResultsWe found a significant (p < 0.05) increase in the percentage of spermatozoa with intact plasma membrane, and intact acrosome in the ZnO (0.1 mg/mL) and Se (1 µg/mL) NP supplemented groups in comparison to the frozen control group. TEM assessment revealed no internalization of both ZnO and Se NPs into the sperm structure. Few occasional contacts of ZnO NPs with the sperm membrane and a few agglomerates of Se NPs around the area of damaged membranes were visualized. HSP70 and HSP90 mRNA levels were significantly (p < 0.001) higher in the NP supplemented groups in comparison to the control. HSP70 and HSP90 mRNA levels had a strong positive association with sperm motility and a weak to moderate association with other sperm parameters. ConclusionsCurrent findings indicated that ZnO NPs are more potent than Se NPs in ameliorating peroxidative damages during sperm cryopreservation, increases semen quality parameters possibly by increasing the expression levels of HSP genes in buck semen. Furthermore, NP supplementation may have a potential role in preserving sperm head ultrastructure by acting as an antioxidant and reducing OS during various degrees of cellular insults, which needs to be further explored.

New pattern of nematode spermatogenesis revealed by ultrastructural observation of Anaplectus granulosus (Plectidae, Plectida)

Summary This paper presents the first comprehensive detailed transmission electron microscope observations of sperm development and structure of a plectid nematode. Sperm development of Anaplectus granulosus resembles that of nematodes of the order Rhabditida, known as the rhabditid pattern of spermatogenesis. It includes formation of complexes of fibrous bodies (FB) with membranous organelles (MO), which appear in spermatocytes; the complexes dissociate in the spermatids. The mature spermatozoa are bipolar cells subdivided into a pseudopod and a main cell body containing a nucleus with nine singlet centrioles, peripheral mitochondria and MOs. However, the development and structure of sperm in A. granulosus deviates remarkably from the common rhabditid pattern by an unusual early transformation of FBs into large amorphous masses in the spermatids; the subsequent formation of a concentric structure of immature spermatozoa with a predominant amorphous mass around the central nucleus and thin peripheral cytoplasm with organelles (MOs and mitochondria); and by the transformation of MO in mature spermatozoa into simple cisterns. Thus, the pattern of spermatogenesis of A. granulosus supports the close relations of Plectida and Rhabditida, but specific peculiarities of the sperm development delineate Plectida from Rhabditida and other orders.

Cryopreservation of equine spermatozoa reduces plasma membrane integrity and phospholipase C zeta 1 content as associated with oocyte activation.

Phospholipase C zeta (PLCZ1) is considered the major sperm-borne oocyte activation factor. Cryopreserved stallion spermatozoa are commonly used for intracytoplasmic sperm injection (ICSI). However, plasma membrane damage and protein modifications caused by cryopreservation could impair sperm structure and function, leading to a reduction of PLCZ1 and oocyte activation after ICSI. We compared membrane integrity and PLCZ1 abundance in populations for fresh, frozen, and refrozen stallion spermatozoa, either thawed and refrozen at room or low temperature; and examined the effect of relative PLCZ1 content on cleavage after ICSI. Western blotting, ELISA, and immunofluorescence were conducted in stallion spermatozoa, freezing extenders, and detergent-extracted sperm fractions to detect and quantify PLCZ1. Retrospectively, PLCZ1 content and cleavage rate were analyzed. Fresh, frozen, and refrozen at room and low temperatures spermatozoa were evaluated for acrosomal and plasma membrane integrity and PLCZ1 content using flow cytometry. Western blotting, ELISA, and immunofluorescence revealed significant reduction of PLCZ1 in spermatozoa after cryopreservation and confirmed PLCZ1 detection in extenders. After detergent extraction, a PLCZ1-nonextractable fraction remained in the postacrosomal region of spermatozoa. Plasma membrane integrity was significantly reduced after freezing. Acrosomal and plasma membrane integrity were similar between frozen and refrozen samples at low temperature, but both were significantly higher than samples refrozen at room temperature. Acrosomal and plasma membrane integrity significantly correlated to PLCZ1 content. Percentages of PLCZ1-labeled spermatozoa and PLCZ1 content were reduced after freezing but not after refreezing. Relative content and localization of PLCZ1 were associated with cleavage rates after ICSI. Sperm PLCZ1 content associates with cleavage rates after ICSI. Cryopreservation is detrimental to sperm plasma membrane integrity and PLCZ1 retention. However, refreezing did not result in additional PLCZ1 loss. Refreezing stallion spermatozoa at a low temperature resulted in better survival but did not improve PLCZ1 retention.

Rapid sperm length divergence in a polygynandrous passerine: a mechanism of cryptic speciation?

When populations become geographically isolated, they begin to diverge in various traits and at variable rates. The dynamics of such trait divergences are relevant for understanding evolutionary processes such as local adaptation and speciation. Here we examine divergences in sperm and body structures in a polygynandrous songbird, the alpine accentor (Prunella collaris) between two allopatric high-altitude populations, in Morocco and Spain. The populations diverged around 82,000 years ago, as estimated with a coalescence-based phylogenetic analysis of genome-wide single-nucleotide polymorphisms. We found that birds in the two areas had nonoverlapping sperm lengths, which suggests adaptation to divergent female reproductive tract environments. Sperm length also showed an exceptionally low coefficient of among-male variation, a signal of strong stabilizing selection imposed by sperm competition. The evolutionary rate of sperm length was almost twice the rates for the most divergent morphological traits and more than three times higher than expected from literature data over a similar generational timescale. This rapid evolution of a key reproductive trait has implications for reproductive isolation and ultimately for speciation. Strong selection for different sperm length optima in allopatry predicts conspecific sperm precedence and disruptive selection in sympatry, hence a possible postcopulatory prezygotic barrier to gene flow.

Analysis of changes in the morphological structures of sperm during preservation of liquid boar semen in two different seasons of the year

Animal fertility is the result of a combination of genetic, physiological and environmental factors. Assessment of semen quality plays a key role in determining the reproductive performance of boars. The aim of the study was to determine the effect of two seasons of the year on the morphology, morphometry, cell membrane integrity and mitochondrial activity of sperm during storage of liquid boar semen at 17 °C. The study was carried out using 20 boars. Four ejaculates were collected from each boar in each of two seasons of the year. All tests were carried out five times: at 1, 24, 48, 96 and 168 h during storage. The results showed that sperm in ejaculates collected in summer are more sensitive to storage conditions than those from ejaculates obtained in winter. The percentage of sperm with morphological defects was shown to increase with the storage time of the diluted ejaculates, particularly between 96 and 168 h of preservation. In summer, the percentage of sperm with an intact cell membrane and the percentage with high mitochondrial membrane potential are much lower than in winter, at every hour of semen preservation. In the case of boars used for artificial insemination, it is worth taking into account the season when the semen is collected, especially if it is to be stored in liquid form. Assessment of sperm cell structures during storage of liquid semen should be implemented at insemination stations and should be carried out more often in summer.

Loss of PMFBP1 Disturbs Mouse Spermatogenesis by Downregulating HDAC3 Expression.

Polyamine modulating factor 1 binding protein (PMFBP1) acts as a scaffold protein for the maintenance of sperm structure. The aim of this study was further to identify the new role and molecular mechanism of PMFBP1 during mouse spermatogenesis. We identified a profile of proteins interacting with PMFBP1 by immunoprecipitation combined with mass spectrometry and demonstrated that class I histone deacetylases, particularly HDAC3 and chaperonin-containing TCP1 subunit 3 (CCT3), were potential interaction partners of PMFBP1 based on network analysis of protein-protein interactions and co-immunoprecipitation. Immunoblotting and immunochemistry assays showed that loss of Pmfbp1 would result in a decline in HDACs and change the proteomic profile of mouse testis, in which differently expressed proteins are associated with spermatogenesis and assembly of flagella, which was proved by proteomic analysis of testis tissue obtained from Pmfbp1-/- mice. After integrating with transcriptome data for Hdac3-/- and Sox30-/- round sperm obtained from a public database, RT-qPCR confirmed ring finger protein 151 (Rnf151) and ring finger protein 133 (Rnf133) were key downstream response factors of the Pmfbp1-Hdac axis affecting mouse spermatogenesis. Taken together, this study indicates a previously unidentified molecular mechanism of PMFBP1 in spermatogenesis whereby PMFBP1 interacts with CCT3, affecting the expression of HDAC3, followed by the downregulation of RNF151 and RNF133, resulting in an abnormal phenotype of sperm beyond the headless sperm tails. These findings not only advance our understanding of the function of Pmfbp1 in mouse spermatogenesis but also provide a typical case for multi-omics analysis used in the functional annotation of specific genes.

The reproductive and transgenerational toxicity of microplastics and nanoplastics: A threat to mammalian fertility in both sexes.

Microplastics (MPs) and nanoplastics (NPs) are extensively distributed in the environment. However, a comprehensive review and in-depth discussion on the effects of MPs and NPs to reproductive capacity and transgenerational toxicity on mammals, especially on humans, is lacked. It is suggested that microplastics and nanoplastics could accumulate in mammalian reproductive organs and exert toxic effects on the reproductive system for both sexes. For males, the damage of microplastics consists of abnormal testicular and sperm structure, decreased sperm vitality, and endocrine disruption, which were caused by oxidative stress, inflammation, apoptosis of testicular cells, autophagy, abnormal cytoskeleton, and abnormal hypothalamic-pituitary-testicular axis. For females, the damage of microplastics includes abnormal ovary and uterus structure and endocrine disruption, which were caused by oxidative stress, inflammation, granulosa cell apoptosis, hypothalamic-pituitary-ovary axis abnormalities, and tissue fibrosis. For transgenerational toxicity, premature mortality existed in the rodent offspring after maternal exposure to microplastics. Among the surviving offspring, metabolic disorders, reproductive dysfunction, immune, neurodevelopmental, and cognitive disorders were detected, and these events directly correlated with transgenerational translocation of MPs and NPs. Studies on human-derived cells or organoids demonstrated that transgenerational toxicity studies for both sexes are yet in the phase of exploring suitable experimental models, and more detailed research on the threat of MPs and NPs to human fertility is still urgently needed. Further studies will help assess the MPs and NPs threat to public fertility and reproductive health risks.

Sperm ultrastructure of Pycnoderes incurvus (Hemiptera: Miridae).

The sperm morphology can provide helpful information about sexual selection, phylogeny, and the evolutionary history of a given animal group. However, there is limited or no knowledge of many taxa, especially those belonging to insects, a vast and highly diverse group. An example is the Miridae, or plant bugs, which belong to the infraorder Cimicomorpha (Heteroptera), where only three out of 17 families have published data on their sperm morphology. Here we described the Miridae sperm structure by analyzing Pycnoderes incurvus sperm under light and transmission electron microscopy. In this species, the spermatozoa were as long and slender as those of most insects. However, the anterior-most region was twisted, a characteristic first reported for Heteroptera. The acrosome was coated with electron-dense material, most likely extra-acrosomal. The centriole adjunct was a notably long, cylindrical and compact structure connecting the nucleus to the flagellar elements, with just clove-like electron-lucent points in cross-section, also features unique to Miridae so far. The flagella exhibited an axoneme of 9 + 9 + 2 microtubules and two symmetrical mitochondrial derivatives. The latter two partially embrace the axoneme, and each exhibits two paracrystalline areas and a bridge connecting it to the axoneme; these are considered Heteroptera synapomorphies that support their monophyly. RESEARCH HIGHLIGHTS: The P. incurvus sperm showed a twisted acrosome, the first reported for Heteroptera. The centriolar adjunct is a sole structure linking the nucleus and flagellum. The flagella presented the synapomorphies supporting Heteroptera monophyly.

Mechanisms of damage to sperm structure in mice on the zinc-deficient diet

BackgroundZinc (Zn)is an essential trace element for spermatogenesis and its deficiency causes abnormal spermatogenesis. ObjectiveThe present study was conducted to examine the mechanisms by which Zn-deficient diet impairs sperm morphology and its reversibility. Methods30 SPF grade male Kunming (KM) mice were randomly divided into three groups, 10 mice per group. Zn-normal diet group (ZN group) was given Zn-normal diet（Zn content= 30 mg/kg）for 8 weeks. Zn-deficienct diet group (ZD group) was given Zn-deficienct diet（Zn content< 1 mg/kg）for 8 weeks. Zn-deficient and Zn-normal diet group（ZDN group）was given 4 weeks Zn-deficienct diet followed by 4 weeks Zn-normal diet. After 8 weeks, the overnight fasted mice were sacrificed, and blood and organs were collected for further analysis. ResultsThe experimental results showed that Zn-deficienct diet leads to increased abnormal morphology sperm and testicular oxidative stress.The rate of abnormal morphology sperm, chromomycin A3(CMA3), DNA fragmentation index (DFI), malondialdehyde (MDA) were significantly increased, and a-kinase anchor protein 4(AKAP4), dynein axonemal heavy chain 1(DNAH1), sperm associated antigen 6(SPAG6), cilia and flagella associated protein 44(CFAP44), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), nuclear factor erythroid 2-related factor (NRF2), NAD(P)H:quinone oxidoreductase 1(NQO1)and heme oxygenase 1(HO1) were significantly decreased in the ZD group mice. While the changes in above indicators caused by Zn-deficient diet were significantly alleviated in the ZDN group. ConclusionIt was concluded that Zn-deficient diet causes abnormal morphology sperm and testicular oxidative stress in male mice. Abnormal morphology sperm caused by Zn-deficient diet are reversible, and Zn-normal diet can alleviate them.

P-103 Three-dimensional oviductal organoid model to study spermatozoa-oviduct interaction in humans

Abstract Study question Is it possible to establish an in vitro model that recapitulating human oviduct to investigate sperm-oviduct interaction Summary answer Three-dimensional (3D) oviductal organoid model was established from either primary human oviductal tissues or immortalized human OE-E6/67 cell line, based on our protocol. What is known already The sperm reservoir in oviduct is formed to maintain the fertilizing capacity of spermatozoa until ovulation by the binding of sperm to the epithelial lining of the oviduct, which is also significant to select sperm with normal chromatin structure and superior fertilization ability. The regulation and mechanism of sperm reservoir formation in human is still unknown, mainly due to the ethical issue and technical difficulty in obtaining enough human oviduct. We previously demonstrated that sperm fucosyltransferase-5 (sFUT5), a carbohydrate-binding protein on spermatozoa, is responsible for spermatozoa-oviduct interaction in humans. Study design, size, duration Human oviductal epithelial cell line immortalized by HPV 16 E6/E7 open reading frame by retroviral expression system was employed to establish the co-culture model. Fresh human samples (human spermatozoa,N = 30; 1-2 cm primary oviductal tissue,N = 15) were collected in hospital with signed consent. Fluorescent pre-labelled sperm were co-cultured with organoids for 2 hours. At least three replicates were conducted for each assay. Participants/materials, setting, methods Morphological characterization and markers expressions of the organoid were determined by immunohistochemistry and confocal microscopy analysis. The sperm binding capacity of the oviductal organoid was studied by co-culture system. Sperm chromatin structure of the organoid-bound sperm was assessed by flow cytometry-based TUNEL assay. Affinity chromatography followed by nano-liquid chromatography–mass spectrometry was used to identify the sFUT5-binding proteins on ovidutal epithelial cells (OECs) and their expressions on OECs were validated by immunostaining/flow cytometry. Main results and the role of chance The organoid exhibited a hollow structure surrounded by a single layer of OECs. Immunohistochemistry analysis revealed the high expressions of epithelial marker E-cadherin (basolateral membrane), secretory marker pair-box 8 (PAX8) and proliferative marker Ki67. Expression of ciliated cell marker, acetylated tubulin (AcTUB) can also be detected. The modification of culture environment successfully reversed the OEC organoids polarity as demonstrated by immunostaining using antibodies against ZO-1 (apical marker) and E-cadherin (basolateral marker), making the organoid accessible during the co-culture. These apical-out OEC organoids have significantly higher sperm binding capacities than their basal-out counterparts. Furthermore, OEC organoid-bound spermatozoa possessed more intact DNA than the unbound spermatozoa. The results indicate the relevance of using OEC organoid-spermatozoa co-culture model as a tool to assess spermatozoa-oviduct interaction in humans. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was then identified as a potential sFUT5-interacting protein on OECs. This result was further supported by flow cytometry and immunofluorescent staining. 3-D oviductal organoid model demonstrates profound potential for in vitro investigation of human oviduct physiology. Limitations, reasons for caution Our 3D model may not fully represent the entire cellular complexity and tubular architecture of the oviductal epithelium. Further investigation on the sperm morphology and DNA integrity and analysis on organoid transcriptome will enhance our understanding on oviduct in human reproduction. Wider implications of the findings The results from co-culture may give a clue to future study on whether oviductal organoid can be applied clinically for functional sperm selection in in vitro fertilization. Our apical-out organoid model may also be applied to study tubal ectopic pregnency. Trial registration number not applicable

The effect of controlled stress on kinematic parameters of cryopreserved stallion sperm

Semen cryopreservation is an important tool for genetic improvement of horse breeds and the reproductive efficiency of stallions. The freezing and thawing process can cause injuries and damage to sperm structures which may impair fertility. The use of controlled stress has been shown to be an alternative for cellular protection against cryoinjuries (Priebenszky et al., Biology of Reproduction. 2010;83:690-697). Thus, this study aimed to evaluate whether the submission of sperm to controlled stress using different negative pressures can improve sperm kinetics. Four healthy stallions aged between 6 and 20 years old were used. Semen was collected (n = 12 ejaculates) and extended with a skim-milk based extender (Botusemen, Botupharma) 1:1 and then transported to the laboratory in a cooling device (Botuflex, Botupharma). The negative pressure exposure technique followed the protocol described by Casali et al., (Acta Scientiae Veterinariae. 2014; 42: 1240) adapted using a vacuum pump (Vix® ¼ hp 60Hz) coupled to a pressure chamber. Each ejaculate was divided and exposed to 4 treatments (Control - no pressure; P200 - submission to a negative pressure of 200 mBar/5min; P500 - negative pressure of 500 mBar/5min; P800 - negative pressure of 800 mBar/5min), centrifuged at 600 gfor 10 min and the pellet was cryopreserved with Botucrio (Botuphatma) at 100 million/ml. For the evaluation of sperm kinetics, computer-assisted sperm analysis (CASA; Androvision software; Minitube) was used. Data were analyzed by ANOVA (SPSS statistical package) followed by Tukeytest (p<0.05). Total motility (Control: 45.9±14.1%; P200: 43.18±10.8%; P500: 45±12.1%; and P800: 41.2±13.2%), circular motility (Control: 1.9±1.2%; P200: 2.3±0.9%; P500: 2.4±1.0%; P800: 1.6± 1.2%), rapid motility (Control: 6.7± 3.7%; P200: 5.5±1.8%; P500: 6.0±4.2%; P800: 4.1±2.3%), slow motility (Control: 20.3±10.2%; P200: 17.4±6.4%; P500: 16.8±7.4%; P800: 15.3±8.9%),non-progressive motility (Control: 16.8±6.5%; P200: 17.9±5.9%; P500: 18.2 ±6.6%; P800: 19.9±8.1%) and progressive motility (Control: 28.7±13.6%; P200: 23.7±9.2%; P500: 26.3±9.5%; P800: 20.7±10.7%) were similar between negative pressure treated and control groups. These findings suggest that negative pressure does not affect sperm motility. However, the effect of negative pressures with the aim to protect sperm against cryo-injures requires further exploration. In conclusion, submission of equine sperm to a controlled stress using different negative pressures does not impair sperm kinetics.

SPAG6 c.900 T>C affects boar semen quality and blood-testis barrier function by creating a new splice acceptor site.

Sperm associated antigen 6 (SPAG6) acts as a scaffolding protein in the center of the flagellar axoneme and has an impact on the maturation of the motility of mammalian sperm flagella and the maintenance of sperm structure. In our previous research, SPAG6 c.900 T>C in exon 7 and exon 7 skipped transcript was identified by analyzing RNA-seq data of testicular tissues from 60 day (sexually immature) and 180 day (sexually mature) Large White boars. Herein, we found porcine SPAG6 c.900 T>C to be associated with semen quality traits in Duroc, Large White and Landrace pigs. SPAG6 c.900 C can generate a new splice acceptor site, inhibit the occurrence of SPAG6 exon 7 skipping to a certain extent, thereby promote the growth of Sertoli cells and maintain the normal blood-testis barrier function. This study provides new insights into the molecular regulation of spermatogenesis and a new genetic marker for the improvement of semen quality in pigs.

Human Sperm as an In Vitro Model to Assess the Efficacy of Antioxidant Supplements during Sperm Handling: A Narrative Review.

Spermatozoa are highly differentiated cells that produce reactive oxygen species (ROS) due to aerobic metabolism. Below a certain threshold, ROS are important in signal transduction pathways and cellular physiological processes, whereas ROS overproduction damages spermatozoa. Sperm manipulation and preparation protocols during assisted reproductive procedures-for example, cryopreservation-can result in excessive ROS production, exposing these cells to oxidative damage. Thus, antioxidants are a relevant topic in sperm quality. This narrative review focuses on human spermatozoa as an in vitro model to study which antioxidants can be used to supplement media. The review comprises a brief presentation of the human sperm structure, a general overview of the main items of reduction-oxidation homeostasis and the ambivalent relationship between spermatozoa and ROS. The main body of the paper deals with studies in which human sperm have been used as an in vitro model to test antioxidant compounds, including natural extracts. The presence and the synergic effects of different antioxidant molecules could potentially lead to more effective products in vitro and, in the future, in vivo.