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Frontiers in Cell and Developmental... 2023Targeted disruption of the soluble adenylyl cyclase (ADCY10; sAC) gene results in male-specific sterility without affecting spermatogenesis, mating behavior, or...
Targeted disruption of the soluble adenylyl cyclase (ADCY10; sAC) gene results in male-specific sterility without affecting spermatogenesis, mating behavior, or spermatozoa morphology and count; however, it dramatically impairs sperm motility and prevents capacitation. These phenotypes were identified in sperm from sAC null mice surgically extracted from the epididymis and studied . Epididymal sperm are dormant, and never exposed to physiological activators in semen or the female reproductive tract. To study sAC null sperm under conditions which more closely resemble natural fertilization, we explored phenotypes of ejaculated sAC null sperm post-coitally as well as , collected from the female reproductive tract. ejaculated sAC null sperm behaved similarly to epididymal sAC null sperm, except with respect to the physiologically induced acrosome reaction. These studies suggest there is a sAC-independent regulation of acrosome responsiveness induced upon ejaculation or exposure to factors in the female reproductive tract. We also studied the behavior of sAC null sperm post-coitally by taking advantage of transgenes with fluorescently labelled sperm. Transgenes expressing GFP in the acrosome and DsRed2 in the mitochondria located in the midpiece of sperm (DsRed2/Acr3-EGFP) allow visualization of sperm migration through the female reproductive tract after copulation. As previously reported, sperm from wild type (WT) double transgenic mice migrated from the uterus through the uterotubular junction (UTJ) into the oviduct within an hour post-copulation. In contrast, sperm from sAC null double transgenic mice were only found in the uterus. There were no sAC null sperm in the oviduct, even 8 h after copulation. These results demonstrate that sAC KO males are infertile because their sperm do not migrate to the fertilization site.
PubMed: 37152282
DOI: 10.3389/fcell.2023.1134051 -
ELife Apr 2023The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis....
The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from , recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.
Topics: Male; Mice; Animals; Sperm Tail; Seeds; Spermatogenesis; Proteins; Spermatids; Testis; Nuclear Proteins; Phosphoproteins; Cell Cycle Proteins
PubMed: 37083624
DOI: 10.7554/eLife.79620 -
Variation of existence and location of aquaporin 3 in relation to cryoresistance of ram spermatozoa.Frontiers in Veterinary Science 2023Osmotic changes during the process of freeze-thawing involve changes in the location of aquaporins (AQPs) in membrane domains of spermatozoa. Some AQPs, like aquaporin 3...
INTRODUCTION AND OBJECTIVE
Osmotic changes during the process of freeze-thawing involve changes in the location of aquaporins (AQPs) in membrane domains of spermatozoa. Some AQPs, like aquaporin 3 (AQP3), are linked to sperm cryotolerance in the porcine species. Conspicuous individual variability exists between rams and their ejaculates, which may be classified as displaying good freezability (GFE) or poor freezability (PFE), depending on several endogenous and environmental factors. The present work aimed to examine whether differences in freezability could even involve changes in location and expression of AQP3 in ram spermatozoa.
METHODS
Thirty ejaculates from 10 rams (three of each) were evaluated and subsequently classified as GFE ( = 13) or PFE ( = 17) through a principal component analysis (PCA) and k-means cluster analysis. Spermatozoa were examined for the presence, abundance and distribution of AQP3 by western blot and immunocytochemistry, employing a commercial rabbit polyclonal antibody (AQP3 - ab125219).
RESULTS AND DISCUSSION
Although AQP3 was found in the sperm acrosome, midpiece, principal and end piece of the tail in both fresh and after frozen-thawed samples, its highest immunolabeling was found in the mid- and principal piece. In the GFE group, the expression of AQP3 in the mid- and principal piece was greater ( < 0.05) in frozen-thawed samples than in fresh specimens while such differences were not detected in the PFE group. Sperm cryotolerance relates to changes in AQP3 expression and thus AQP3 could be used as a biomarker for cryotolerance.
CONCLUSION
A greater capacity of AQP3 localization in mid- and principal piece of the spermatozoa could be linked to an increase the osmo-adaptative capacity of ejaculates with better capacity to withstand freeze-thawing processes.
PubMed: 37056227
DOI: 10.3389/fvets.2023.1167832 -
International Journal of Molecular... Mar 2023To evaluate whether the follicle-stimulating hormone (FSH) receptor (FSHR) is expressed in human spermatozoa and the effects of FSH incubation on sperm function....
To evaluate whether the follicle-stimulating hormone (FSH) receptor (FSHR) is expressed in human spermatozoa and the effects of FSH incubation on sperm function. Twenty-four Caucasian men were recruited. Thirteen patients had asthenozoospermia, and the remaining 11 had normal sperm parameters (controls). After confirming FSHR expression, spermatozoa from patients and controls were incubated with increasing concentrations of human purified FSH (hpFSH) to reassess FSHR expression and localization and to evaluate progressive and total sperm motility, the mitochondrial membrane potential, and protein kinase B (AKT) 473 and 308 phosphorylation. FSHR is expressed in the post-acrosomal segment, neck, midpiece, and tail of human spermatozoa. Its localization does not differ between patients and controls. Incubation with hpFSH at a concentration of 30 mIU/mL appeared to increase FSHR expression mainly in patients. Incubation of human spermatozoa with hpFSH overall resulted in an overall deterioration of both progressive and total motility in patients and controls and worse mitochondrial function only in controls. Finally, incubation with FSH increased AKT/tubulin phosphorylation to a greater extent than AKT. FSHR is expressed in the post-acrosomal region, neck, midpiece, and tail of human spermatozoa. Contrary to a previous study, we report a negative effect of FSH on sperm motility and mitochondrial function. FSH also activates the AKT signaling pathway.
Topics: Humans; Male; Follicle Stimulating Hormone; Proto-Oncogene Proteins c-akt; Sperm Motility; Semen; Follicle Stimulating Hormone, Human; Receptors, FSH; Spermatozoa
PubMed: 37047508
DOI: 10.3390/ijms24076536 -
Journal of Veterinary Research Mar 2023Bees are currently artificially inseminated on a large scale for breeding and research purposes. The sperm of bees has a complex and varied structure, and determination...
INTRODUCTION
Bees are currently artificially inseminated on a large scale for breeding and research purposes. The sperm of bees has a complex and varied structure, and determination of specific morphological defects in it is very difficult. Its comprehensive analysis by inspecting morphology and morphometry is an important tool for improving honey bee lines. The staining technique should interfere with the cells as little as possible while clearly showing the boundaries of the head and other elements. In this study, a comparative analysis of the morphometry of sperm was performed with various techniques for staining drone semen.
MATERIAL AND METHODS
Semen was collected from 150 sexually mature Buckfast bee drones by artificially everting the copulatory organ. The morphology and morphometry of the sperm were assessed on slides prepared by three staining methods according to the protocols described online, using the Sperm Class Analyzer system. The lengths of the acrosome, nucleus, head in total, midpiece, tail without midpiece, tail with midpiece, and entire sperm were measured.
RESULTS
The most details of the drone sperm structure could be seen when stained with the eosin-nigrosin complex. This method made it possible to identify all structures and revealed the uneven distribution of sperm proteins in different parts of the tail. With the Sperm Stain method fewer details of the sperm structure were recognisable, and the fewest were with SpermBlue.
CONCLUSION
The staining method, and thus the chemical reagents used, affect the dimensions of drone sperm. Given the great research potential of modified spermatozoa of insects, a standard for slide preparation for the evaluation of morphological and morphometric semen parameters should be established, as this would facilitate result comparison between laboratories and increase the value of morphological analysis of sperm for predicting and assessing fertility.
PubMed: 37008773
DOI: 10.2478/jvetres-2023-0001 -
Journal of Personalized Medicine Mar 2023Sperm DNA fragmentation (SDF) levels have been measured in the workup for in vitro fertilization (IVF) at PIVET since 2007, with the Halosperm test having replaced the...
Sperm DNA fragmentation (SDF) levels have been measured in the workup for in vitro fertilization (IVF) at PIVET since 2007, with the Halosperm test having replaced the previous sperm chromatin structure assay (SCSA) since 2013. Of 2624 semen samples analyzed for the Halosperm test, 57 were excluded as the sperm concentration was <5 million/mL, a level too low for accurate testing, leaving 2567 samples for assessment within this study. The SDF rates were categorized in 5 sperm DNA fragmentation indices (DFI), ranging from <5% to levels >30%, and these categories were correlated with the respective semen analysis profiles and two clinical parameters, namely the age of the male and the ejaculatory abstinence period prior to the sample. The results showed a significant correlation with male age (r = 0.088; < 0.0001), the abstinence period (r = 0.076; = 0.0001), and the semen volume (r 0.063; = 0.001), meaning an adversely high SDF was associated with advanced age, prolonged abstinence, and raised semen volume parameters. There was a significant negative correlation with sperm morphology (r = -0.074; = 0.0001), progressive motility (r = -0.257; < 0.0001), and semen pH (r = -0.066; < 0.001), meaning these semen anomalies were associated with high SDF values. With respect to abnormal morphology, sperm tail defects had a positive correlation (r = 0.096; < 0.0001) while midpiece defects showed a negative correlation (r = -0.057; = 0.004), meaning that tail defects are most likely to associate with adverse DFI values. With respect to motility patterns, the poorer patterns showed a positive correlation with increased DFI, namely C pattern (r = 0.055; = 0.005) and D pattern (r = 0.253; < 0.0001). These results imply that raised DFI reflects poor sperm quality and should be investigated in clinical trials involving IVF and the consideration of intracytoplasmic sperm injection (ICSI).
PubMed: 36983700
DOI: 10.3390/jpm13030518 -
Frontiers in Reproductive Health 2023Prior research has substantiated the vital role of telomeres in human fertility. Telomeres are prerequisites for maintaining the integrity of chromosomes by preventing... (Review)
Review
Prior research has substantiated the vital role of telomeres in human fertility. Telomeres are prerequisites for maintaining the integrity of chromosomes by preventing the loss of genetic material following replication events. Little is known about the association between sperm telomere length and mitochondrial capacity involving its structure and functions. Mitochondria are structurally and functionally distinct organelles that are located on the spermatozoon's midpiece. Mitochondria produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS), which is necessary for sperm motility and generate reactive oxygen species (ROS). While a moderate concentration of ROS is critical for egg-sperm fusion, and fertilization, excessive ROS generation is primarily related to telomere shortening, sperm DNA fragmentation, and alterations in the methylation pattern leading to male infertility. This review aims to highlight the functional connection between mitochondria biogenesis and telomere length in male infertility, as mitochondrial lesions have a damaging impact on telomere length, leading both to telomere lengthening and reprogramming of mitochondrial biosynthesis. Furthermore, it aims to shed light on how both inositol and antioxidants can positively affect male fertility.
PubMed: 36890798
DOI: 10.3389/frph.2023.1107215 -
International Journal of Molecular... Feb 2023Porcine spermatozoa are stored in the oviductal isthmus after natural mating, and the number of spermatozoa is increased in the oviductal ampulla when the mature...
Porcine spermatozoa are stored in the oviductal isthmus after natural mating, and the number of spermatozoa is increased in the oviductal ampulla when the mature cumulus-oocyte complexes (COCs) are transferred into the ampulla. However, the mechanism is unclear. Herein, natriuretic peptide type C (NPPC) was mainly expressed in porcine ampullary epithelial cells, whereas its cognate receptor natriuretic peptide receptor 2 (NPR2) was located on the neck and the midpiece of porcine spermatozoa. NPPC increased sperm motility and intracellular Ca levels, and induced sperm release from oviduct isthmic cell aggregates. These actions of NPPC were blocked by the cyclic guanosine monophosphate (cGMP)-sensitive cyclic nucleotide-gated (CNG) channel inhibitor -Diltiazem. Moreover, porcine COCs acquired the ability to promote NPPC expression in the ampullary epithelial cells when the immature COCs were induced to maturation by epidermal growth factor (EGF). Simultaneously, transforming growth factor-β ligand 1 (TGFB1) levels were dramatically increased in the cumulus cells of the mature COCs. The addition of TGFB1 promoted NPPC expression in the ampullary epithelial cells, and the mature COC-induced NPPC was blocked by the transforming growth factor-β type 1 receptor (TGFBR1) inhibitor SD208. Taken together, the mature COCs promote NPPC expression in the ampullae via TGF-β signaling, and NPPC is required for the release of porcine spermatozoa from the oviduct isthmic cells.
Topics: Female; Humans; Male; Swine; Animals; Oocytes; Sperm Motility; Semen; Oviducts; Spermatozoa; Transforming Growth Factors; Natriuretic Peptides
PubMed: 36834527
DOI: 10.3390/ijms24043118 -
Antioxidants (Basel, Switzerland) Feb 2023Mitochondrial uncoupling proteins (UCPs) are central in the regulation of mitochondrial activity and reactive oxygen species (ROS) production. High oxidative stress is a...
Mitochondrial uncoupling proteins (UCPs) are central in the regulation of mitochondrial activity and reactive oxygen species (ROS) production. High oxidative stress is a major cause of male infertility; however, UCPs expression and function in human spermatozoa are still unknown. Herein, we aimed to assess the expression and function of the different homologs (UCP1-6) in human spermatozoa. For this purpose, we screened for the mRNA expression of all UCP homologs. Protein expression and immunolocalization of UCP1, UCP2, and UCP3 were also assessed. Highly motile spermatozoa were isolated from human normozoospermic seminal samples (n = 16) and incubated with genipin, an inhibitor of UCPs (0, 0.5, 5, and 50 µM) for 3 h at 37 °C. Viability and total motility were assessed. Mitochondrial membrane potential and ROS production were evaluated. Media were collected and the metabolic profile and antioxidant potential were analyzed by H-NMR and FRAP, respectively. The expression of all UCP homologs () mRNA by human spermatozoa is herein reported for the first time. UCP1-3 are predominant at the head equatorial segment, whereas UCP1 and UCP2 are also expressed at the spermatozoa midpiece, where mitochondria are located. The inhibition of UCPs by 50 µM genipin, resulting in the UCP3 inhibition, did not compromise sperm cell viability but resulted in irreversible total motility loss that persisted despite washing or incubation with theophylline, a cAMP activator. These effects were associated with decreased mitochondrial membrane potential and lactate production. No differences concerning UCP3 expression, however, were observed in spermatozoa from normozoospermic versus asthenozoospermic men (n = 6). The inhibition of UCPs did not increase ROS production, possibly due to the decreased mitochondrial activity and genipin antioxidant properties. In sum, UCPs are major regulators of human spermatozoa motility and metabolism. The discovery and characterization of UCPs' role in human spermatozoa can shed new light on spermatozoa ROS-related pathways and bioenergetics physiology.
PubMed: 36829970
DOI: 10.3390/antiox12020409 -
Scientific Reports Feb 2023Asthenozoospermia (AZS) is a severe form of male infertility with no clear pathogenesis, despite numerous research efforts, there is no consensus on this. This study was...
Asthenozoospermia (AZS) is a severe form of male infertility with no clear pathogenesis, despite numerous research efforts, there is no consensus on this. This study was to investigate the expression of gene-associated with retinoid-interferon-induced mortality 19 (GRIM-19) in the sperm of patients with asthenozoospermia and the regulation of GC-2 spd cell proliferation, apoptosis and migration. We analyzed the sperm samples from 82 asthenozoospermia and normal patients were collected in the First People's Hospital of Shangqiu and the First Affiliated Hospital of Zhengzhou University. Immunofluorescence, western blots and RT-qPCR analyses were used to verify the expressions of GRIM-19. MTT assays were used to assess cell proliferations, flow cytometry was performed to assess cell apoptosis, wound‑healing was performed to measure cell migration. Immunofluorescence showed that GRIM-19 is predominantly expressed in the sperm mid-piece, the mRNA expressions of GRIM-19 in sperms of the asthenozoospermia group were significantly low, relative to the normal group (OR 0.266; 95% CI = 0.081-0.868; P = 0.028). The protein expressions of GRIM-19 in sperms of the asthenozoospermia group were significantly lower than that of the normal group as well (GRIM-19/GAPDH: 0.827 ± 0.063 vs 0.458 ± 0.033; P < 0.001). GRIM-19 overexpression promotes GC-2 spd cell proliferation and migration and reduces apoptosis, while GRIM-19-silenced reduces GC-2 spd cell proliferation and migration and increased apoptosis. GRIM-19 is closely related to the occurrence of asthenozoospermia and promotes GC-2 spd cell proliferation and migration and reduces apoptosis.
Topics: Humans; Male; Asthenozoospermia; Semen; Apoptosis; Apoptosis Regulatory Proteins; Cell Proliferation
PubMed: 36813832
DOI: 10.1038/s41598-023-29775-7