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Medicina (Kaunas, Lithuania) Oct 2022Background and Objectives: Septins (SEPTs) are highly conserved GTP-binding proteins and the fourth component of the cytoskeleton. Polymerization of SEPTs contributes to...
Background and Objectives: Septins (SEPTs) are highly conserved GTP-binding proteins and the fourth component of the cytoskeleton. Polymerization of SEPTs contributes to several critical cellular processes such as cytokinesis, cytoskeletal remodeling, and vesicle transportation. In our previous study, we found that SEPT14 mutations resulted in teratozoospermia with >87% sperm morphological defects. SEPT14 interactors were also identified through proteomic assays, and one of the peptides was mapped to RAB3B and RAB3C. Most studies on the RAB3 family have focused on RAB3A, which regulates the exocytosis of neurotransmitters and acrosome reactions. However, the general expression and patterns of the RAB3 family members during human spermatogenesis, and the association between RAB3 and teratozoospermia owing to a SEPT14 mutation, are largely unknown. Materials and Methods: Human sperm and murine male germ cells were collected in this study and immunofluorescence analysis was applied on the collected sperm. Results: In this study, we observed that the RAB3C transcripts were more abundant than those of RAB3A, 3B, and 3D in human testicular tissues. During human spermatogenesis, the RAB3C protein is mainly enriched in elongated spermatids, and RAB3B is undetectable. In mature human spermatozoa, RAB3C is concentrated in the postacrosomal region, neck, and midpiece. The RAB3C signals were delocalized within human spermatozoa harboring the SEPT14 mutation, and the decreased signals were accompanied by a defective head and tail, compared with the healthy controls. To determine whether RAB3C is involved in the morphological formation of the head and tail of the sperm, we separated murine testicular tissue and isolated elongated spermatids for further study. We found that RAB3C is particularly expressed in the manchette structure, which assists sperm head shaping at the spermatid head, and is also localized at the sperm tail. Conclusions: Based on these results, we suggest that the localization of RAB3C proteins in murine and human sperm is associated with SEPT14 mutation-induced morphological defects in sperm.
Topics: Mice; Humans; Male; Animals; Teratozoospermia; Septins; Proteomics; Semen; Spermatozoa; GTP-Binding Proteins; Peptides
PubMed: 36295569
DOI: 10.3390/medicina58101408 -
Molecular Human Reproduction Aug 2023Freezing and thawing diminish sperm motility and fertility by disrupting the cholesterol balance in sperm plasma and organelle membranes. The aim of this study was to...
Freezing and thawing diminish sperm motility and fertility by disrupting the cholesterol balance in sperm plasma and organelle membranes. The aim of this study was to elucidate the mechanisms through which exogeneous cholesterol treatment enhances the quality of frozen-thawed bull sperm. The incorporation of cholesterol was investigated using boron-dipyrromethene (BODIPY)-cholesterol, and BODIPY signals were detected not only in the plasma membrane but also in the midpiece region immediately after thawing. The positive signal of cholesterol in the midpiece region was inhibited by a scavenger receptor class B Type I (SR-BI) inhibitor, block lipid transport 1 (BLT-1). To comprehend the role of exogenous cholesterol in the functions of the plasma membrane, propidium iodide (PI)/Annexin V and peanut agglutinin lectin (PNA) staining were performed. The results showed that treatment with exogenous cholesterol increased the number of acrosome-intact sperm and decreased the number of sperm with damage to the plasma membrane. Moreover, since BODIPY signals were also observed in the midpiece region, mitochondrial function was evaluated using a flux analyzer and a flow cytometer with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) staining, revealing an increase in the number of sperm with high-mitochondrial activity and oxygen consumption. Finally, to assess sperm fertility, computer-assisted sperm analysis (CASA) and IVF were carried out. Sperm velocities and fertilization rates in IVF were significantly enhanced by the addition of cholesterol just after thawing. Thus, the treatment with cholesterol after thawing protected the plasma membrane from the stress of thawing and maintained mitochondrial function, thereby preserving the fertilization ability of frozen-thawed bull sperm for conventional IVF and artificial insemination (AI). Therefore, the application of cholesterol just after thawing is a promising option for improving the fertility of frozen-thawed sperm.
Topics: Male; Animals; Cattle; Semen; Sperm Motility; Spermatozoa; Cholesterol; Fertility
PubMed: 37656939
DOI: 10.1093/molehr/gaad031 -
Reproductive Medicine and Biology 2022In humans, catecholamines (including dopamine) have been identified in semen and fallopian tubes, while dopamine D2 receptors (D2DR) are found in the sperm midpiece...
PURPOSE
In humans, catecholamines (including dopamine) have been identified in semen and fallopian tubes, while dopamine D2 receptors (D2DR) are found in the sperm midpiece region. How dopamine dose affects human sperm function and whether dopamine treatment is useful in assisted reproductive technology is unclear.
METHODS
Sperm samples were obtained from patients with normal semen parameters undergoing fertility treatment. We investigated the effects of dopamine treatment on tyrosine phosphorylation and sperm motility. Sperm motility was analyzed using the computer-assisted sperm analysis (CASA) system.
RESULTS
This study revealed that various dopamine concentrations (0.1-100 μM) did not increase sperm tyrosine phosphorylation. Progressive motility increased substantially when treated with high concentrations of dopamine (10 and 100 μM) and was blocked by raclopride (a D2DR antagonist). After 24-h sperm culture, the addition of 10 μM dopamine significantly increased curvilinear velocity and amplitude of lateral head displacement, which are indicators of hyperactivation.
CONCLUSION
Dopamine did not affect tyrosine phosphorylation, but increased sperm motility. High concentrations of dopamine were more effective to accelerate sperm motility in cases where sperm motile capacity was low.
PubMed: 36310655
DOI: 10.1002/rmb2.12482 -
Reproduction in Domestic Animals =... Dec 2019Sperm morphometry is the tool that confers objectivity to the morphological evaluation by accurately measuring the dimensions of the gamete and its structures. Thus, the...
Sperm morphometry is the tool that confers objectivity to the morphological evaluation by accurately measuring the dimensions of the gamete and its structures. Thus, the aim of the study was to perform a morphometric characterization of the domestic cat sperm. Therefore, sperm samples were collected from twenty pairs of epididymis in a TRIS extender at 37ºC. An aliquot of the sample was used to make a smear with Rose Bengal solution, and afterwards, the morphology and morphometry were analysed. In the morphology, were quantified the percentage of normal sperm cells, morphological changes of head, midpiece and tail. In morphometry, each normal sperm cell was measured for length, width, area and perimeter of head and midpiece, tail length and total length. The parameters ellipticity, elongation, regularity and rugosity were also determined. The percentage of normal sperm was 67.21%. Of the abnormalities, the curled/folded tail, followed by the curved midpiece, abnormal shaped head and detached head were the most quantified. The sperm head presented 5.56 ± 0.01 μm and 3.10 ± 0.01 μm of length and width, respectively. The head area was 16.94 ± 0.05 μm , while the perimeter was 16.16 ± 0.03 μm. In the derived parameters, the values were as follows: ellipticity of 1.81 ± 0.00; elongation of 21.39 ± 0.12; regularity of 0.81 ± 0.00; and rugosity of 0.14 ± 0.00. The midpiece presented length and width of 7.96 ± 0.01 μm and 0.76 ± 0.01 μm, respectively. The mean length of the sperm tail was 45.12 ± 0.06 μm, and the total cell size was 58.67 ± 0.06 μm. Thus, it was concluded that the cat sperm is an elongated cell, with high rugosity and regularity. The spermatic tail represents more than ¾ of the total length of the cell and the midpiece exceeds the length of the head.
Topics: Animals; Cats; Epididymis; Image Processing, Computer-Assisted; Male; Semen Analysis; Software; Sperm Head; Sperm Tail; Spermatozoa
PubMed: 31573695
DOI: 10.1111/rda.13572 -
Clinical and Experimental Reproductive... Jun 2021Bacteriospermia and urogenital infections are common problems in male infertility. This study aimed to evaluate the effects of bacteriospermia on sperm parameters and...
OBJECTIVE
Bacteriospermia and urogenital infections are common problems in male infertility. This study aimed to evaluate the effects of bacteriospermia on sperm parameters and clinical outcomes in semen samples infected with two common bacteria (Staphylococcus saprophyticus and Escherichia coli) in northern Iran.
METHODS
Microbiological tests were performed to isolate and identify organisms from 435 semen samples from infertile couples. Semen samples were assessed according to the World Health Organization criteria. The protamine status, chromatin structure, chromatin condensation, and acrosome reaction of sperm and assisted reproductive outcomes were determined in couples with different male infertility factors.
RESULTS
Among the total cases, the two most prevalent pathogens were considered: S. saprophyticus (38.2%) and E. coli (52.9%). In the semen samples infected with E. coli, the spontaneous acrosome reaction and abnormal chromatin condensation were more common (p<0.05). Significant increases in abnormal chromatin condensation and deprotamination were seen in the presence of S. saprophyticus. In washed semen, tight adhesion between the sperm midpiece and S. saprophyticus was observed. There was also a significant decrease in the fertilization rate using semen samples infected with S. saprophyticus and E. coli during in vitro fertilization cycles (p<0.001). In addition, the presence of S. saprophyticus and E. coli in semen samples was associated with a lower likelihood of clinical pregnancy in couples with various factors of male infertility.
CONCLUSION
Poor results of assisted reproductive techniques may be correlated with semen samples infected with two common bacteria in northern Iran.
PubMed: 34078007
DOI: 10.5653/cerm.2020.04203 -
Andrologia Mar 2022Human semen is a heterogeneous group containing a portion of low-motility sperm, which may determine the sperm quality evaluation. Abnormally expressed proteins in...
Human semen is a heterogeneous group containing a portion of low-motility sperm, which may determine the sperm quality evaluation. Abnormally expressed proteins in low-motility spermatozoa will be the candidates for sperm biology research. By comparing proteomes of high- or low-motility spermatozoa from the same semen of normal fertile men, 21 differentially expressed proteins were identified. Proteins with molecular chaperone function were significantly over-represented, of which HSPA1L and HSPA9 significantly decreased in low-motility sperm. Compared with young adult testes with normal spermatogenesis, HSPA1L and HSPA9 had decreased expressions in elderly testis characterised with poor spermatogenesis, suggesting their associations with spermatogenesis. Decreased expressions of HSPA1L and HSPA9 in low-motility spermatozoa were validated by Western Blot and immunofluorescence quantification analysis. HSPA1L was mainly expressed on sperm post-acrosome and midpiece, whilst HSAP9 was mainly expressed on acrosome and sperm tail. HSPA1L antibody could inhibit sperm motility validated by antibody blocking experiment, whilst HSPA9 antibody showed no significant effect on sperm motility. The study demonstrated that low-motility spermatozoa from fertile men had poor sperm quality, in which differential expressed proteins were promising markers for evaluating sperm quality, understanding mechanism of male infertility with unexplained causes, and providing new idea for male infertility research.
Topics: Aged; Fertility; HSP70 Heat-Shock Proteins; Humans; Infertility, Male; Male; Mitochondrial Proteins; Semen Analysis; Sperm Motility; Spermatogenesis; Spermatozoa; Young Adult
PubMed: 34796524
DOI: 10.1111/and.14321 -
Metformin improves fish sperm quality by regulating glucose uptake capacity during in vitro storage.Journal of Animal Science Jan 2023A suitable additive for fish sperm storage in vitro is necessary for artificial reproduction. In this study, we evaluated the effects of different concentrations (100,...
A suitable additive for fish sperm storage in vitro is necessary for artificial reproduction. In this study, we evaluated the effects of different concentrations (100, 200, 400, and 800 µmol/L) of metformin (Met) on Schizothorax prenanti and Onychostoma macrolepis sperm under storage in vitro for 72 h. Compared with the control group, 400 µmol/L Met was more effective at improving the quality and fertilization capacity of S. prenanti sperm by increasing the adenosine triphosphate (ATP) content within the sperm. Further study found that Met stabilized the ATP level by enhancing the glucose uptake in S. prenanti sperm, and this effect might be associated with the activation of AMP-activated protein kinase (AMPK) in sperm. In this study, we also found that glucose could be absorbed by the sperm of S. prenanti, which was mainly accumulated in the midpiece of S. prenanti sperm, where mitochondria were located. In addition, Compound C significantly inhibited the beneficial effects of Met on the quality and glucose uptake capacity of S. prenanti sperm by inhibiting AMPK phosphorylation. These results revealed that AMPK played an important role in vitro sperm storage, and Met maintained ATP content and increased the storage time of S. prenanti sperm in vitro for 72 h, possibly due to Met enhanced glucose uptake capacity of sperm by activating AMPK. Similarly, the beneficial effects of Met on S. prenanti sperm were also found in O. macrolepis sperm, suggesting that Met may hold great promise for the practice of storing fish in vitro.
Topics: Male; Animals; Metformin; AMP-Activated Protein Kinases; Semen; Spermatozoa; Cyprinidae; Adenosine Triphosphate
PubMed: 37191447
DOI: 10.1093/jas/skad152 -
Human Reproduction (Oxford, England) May 2022Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca2+-signalling and function?
STUDY QUESTION
Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca2+-signalling and function?
SUMMARY ANSWER
While APAP itself does not interact with Ca2+-signalling in human sperm, its metabolite N-arachidonoyl phenolamine (AM404), produced via fatty acid amide hydrolase (FAAH), interferes with human sperm Ca2+-signalling and function through a suggested CatSper channel-dependent action.
WHAT IS KNOWN ALREADY
Studies have shown that adult men with high urinary levels of over-the-counter mild analgesic APAP have impaired sperm motility and increased time-to-pregnancy.
STUDY DESIGN, SIZE, DURATION
This study consists of (i) an in vivo human pharmaceutical APAP exposure experiment to understand to what degree APAP reaches the sperm cells in the seminal fluid; (ii) in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and (iii) experiments to understand the in situ capabilities of human sperm cells to form APAP metabolite AM404.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Three healthy young males participated in the in vivo human exposure experiment after prior consent. Human semen samples were provided by healthy young volunteer donors after prior consent on the day of the in vitro experiments.
MAIN RESULTS AND THE ROLE OF CHANCE
Pharmaceutical APAP exposure reaches the seminal plasma in high micromolar concentrations and accumulates in the seminal plasma between 3 and 5 days of exposure (P-value 0.023). APAP and its primary metabolite 4-aminophenol (4AP) do not interact with human sperm Ca2+-signalling. Instead, the APAP metabolite AM404 produced via FAAH interferes with human sperm Ca2+-signalling through a CatSper-dependent action. Also, AM404 significantly increases sperm cell penetration into viscous mucous (P-value of 0.003). FAAH is functionally expressed in human sperm cells in the neck/midpiece region, as evidenced by immunohistochemical staining and the ability of human sperm cells to hydrolyse the fluorogenic FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in an FAAH-dependent manner. Importantly, human sperm cells have the capacity to form AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells).
LIMITATIONS, REASONS FOR CAUTION
The experiments were conducted largely in vitro. Future studies are needed to test whether APAP can disrupt human sperm function in vivo through the action of AM404.
WIDER IMPLICATIONS OF THE FINDINGS
We hypothesize that these observations could, at least in part, be responsible for the negative association between male urinary APAP concentrations, sperm motility and time-to-pregnancy.
STUDY FUNDING/COMPETING INTEREST(S)
D.M.K. is funded by the Lundbeck Foundation, grant number R324-2019-1881, and the Svend Andersen Foundation. A.R. is funded by a BRIDGE-Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF18SA0034956. All authors declare no competing interests.
TRIAL REGISTRATION NUMBER
N/A.
Topics: Acetaminophen; Adult; Arachidonic Acids; Calcium; Calcium Channels; Humans; Male; Pharmaceutical Preparations; Progesterone; Sperm Motility; Spermatozoa
PubMed: 35259261
DOI: 10.1093/humrep/deac042 -
Nature Communications Nov 2022Environmental change frequently drives morphological diversification, including at the cellular level. Transitions in the environment where fertilization occurs (i.e.,...
Environmental change frequently drives morphological diversification, including at the cellular level. Transitions in the environment where fertilization occurs (i.e., fertilization mode) are hypothesized to be a driver of the extreme diversity in sperm morphology observed in animals. Yet how fertilization mode impacts the evolution of sperm components-head, midpiece, and flagellum-each with different functional roles that must act as an integrated unit remains unclear. Here, we test this hypothesis by examining the evolution of sperm component lengths across 1103 species of vertebrates varying in fertilization mode (external vs. internal fertilization). Sperm component length is explained in part by fertilization mode across vertebrates, but how fertilization mode influences sperm evolution varies among sperm components and vertebrate clades. We also identify evolutionary responses not influenced by fertilization mode: midpieces evolve rapidly in both external and internal fertilizers. Fertilization mode thus influences vertebrate sperm evolution through complex component- and clade-specific evolutionary responses.
Topics: Animals; Male; Biological Evolution; Semen; Spermatozoa; Vertebrates; Fertilization
PubMed: 36357384
DOI: 10.1038/s41467-022-34609-7 -
Scientific Reports Mar 2022Digital holographic microscopy (DHM) was applied for the morphological assessment of live intact spermatozoa from fertile and infertile men directly after semen...
Digital holographic microscopy (DHM) was applied for the morphological assessment of live intact spermatozoa from fertile and infertile men directly after semen liquefaction. This method allowed us to study the sperm population directly from the sample droplet and not only from the focal plane of the microscope as in classical optical microscopy. The newly implemented 3-dimensional sperm morphological parameters (head height, acrosome/nucleus height, head/midpiece height) were included in morphological assessment of semen samples from fertile and infertile individuals. The values of the 3D parameters were less variable in fertile men than for infertile ones. DHM was also used to compare the morphological profiles of spermatozoa after applying the "swim-up" and gradient centrifugation techniques. During selection, the most statistically significant differences were observed after separation with a Percoll gradient of 90% and a 60-min "swim-up" procedure versus 'native' unfractionated samples. This shows that the developed methodology can be efficiently used for the selection of morphologically sound spermatozoa. The motility type for each spermatozoon was also assessed. The results indicate that the extension of the number of morphological parameters with new 3D parameters and the simultaneous assessment of sperm motility may be valuable addition to sperm examination.
Topics: Acrosome; Humans; Male; Microscopy; Semen Analysis; Sperm Motility; Spermatozoa
PubMed: 35318373
DOI: 10.1038/s41598-022-08798-6