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Fertility and Sterility Apr 2023To obtain de novo male gametes capable of inducing full preimplantation blastocyst development using a novel three-dimensional (3D) culture system.
OBJECTIVE
To obtain de novo male gametes capable of inducing full preimplantation blastocyst development using a novel three-dimensional (3D) culture system.
DESIGN
Mouse embryonic stem cells (mESCs) were spherified by plunging in sodium alginate followed by calcium chloride, delineating a 3D environment that simulates the seminiferous tubule. As a control, mESCs cultured on two-dimensional plates were used. Plates and spheres containing mESCs from both methods were exposed to Activin-A, bFGF, and KSR followed by exposure to BMP4, LIF, SCF, and EGF to promote differentiation into male germ-like cells.
MAIN OUTCOME MEASURES
Cells were assessed for VASA, DAZL, and BOULE on days 3 and 10. Cells were later injected into activated oocytes and monitored using time-lapse imaging on days 15, 22, 29, and 36. Control conceptuses generated using mature epididymal spermatozoa were also monitored via time-lapse imaging.
RESULTS
On day 3, cells differentiated on plates expressed VASA at 1% and DAZL at 29%. In spheres, VASA was expressed at a rate of 15% and DAZL at a rate of 45% (P<.001). On day 10, cells differentiated on plates had VASA expression of 7%, DAZL of 23%, and BOULE of only 0.5%. Cells differentiated into spheres expressed VASA at a rate of 20%, DAZL at 43%, and BOULE at 10% (P<.001). Subsequent differentiation in spheres on day 3 exhibited a DAZL (expressed in spermatogonia) expression of 43% and a VASA (further spermatogenesis progression) expression of 15%. On day 10, DAZL and VASA expressions were reassessed and increased to 45% and 18%, respectively. BOULE, a marker expressed solely in postmeiotic spermatocytes, was expressed at 8%, whereas acrosin was expressed in spermatids at 2%. On day 15, VASA expression plateaued at 17%, BOULE peaked at 10%, and acrosin reached 5%. On day 22, expression of VASA increased to 19%, BOULE decreased to 8%, and acrosin peaked at 7%. On day 29, VASA expression peaked at 20%, BOULE dropped to 2%, and acrosin remained stable at 7%. On day 36, VASA expression remained at 13%, whereas BOULE and acrosin expression decreased to 0% and 1%, respectively. The control cohort attained 88.4% fertilization and 76.9% blastocyst rates. De novo gametes achieved fertilization rates of 35.0%, 61.1%, 81.8%, and 50.0% on days 15, 22, 29, and 36, respectively. Neogametes-generated blastocyst rates were 5.0%, 16.7%, 36.4%, and 8.3% for days 15, 22, 29, and 36, respectively.
CONCLUSION
Our novel 3D differentiation model can generate functional gametes and is aimed at obviating the need for allogeneic/xenogeneic transplantation. The decreased overall marker expression and the reduced blastocyst development indicated that intrasphere germ cell differentiation correlated with the length of mouse spermatogenesis at approximately 30 days. Future experiments will be conducted to confirm the reproducibility of our findings and the eventual generation of offspring.
Topics: Male; Animals; Mice; Acrosin; Haploidy; Reproducibility of Results; Spermatozoa; Spermatogenesis; Spermatocytes
PubMed: 36706828
DOI: 10.1016/j.fertnstert.2023.01.021 -
Andrology Oct 2023Previously, we reported that cysteine-rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine-rich secretory protein...
BACKGROUND
Previously, we reported that cysteine-rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine-rich secretory protein 2protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning.
OBJECTIVES
This study aimed to identify cysteine-rich secretory protein 2 interacting partners. These binding partner interactions were investigated under different conditions, namely, non-capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187-induced acrosome reaction.
MATERIALS AND METHODS
The incubated pig sperm samples were subjected to protein extraction. Extracted proteins were subjected to blue native gel electrophoresis and native immunoblots. Immunoreactive gel bands were excised and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC-MS for protein identification. The most prominent cystein-rich secretory protein 2 interacting proteins that appeared in both independent LC-MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with cystein-rich secretory protein 2 in pig sperm.
RESULTS
Blue native gel electrophoresis and native immunoblots revealed that cystein-rich secretory protein 2 was present within a ∼150 kDa protein complex under all three conditions. Interrogation of cystein-rich secretory-protein 2-immunoreactive bands from blue native gels as well as cystein-rich secretory protein 2 immunoprecipitated products using mass spectrometry consistently revealed that, beyond cystein-rich secretory protein 2, acrosin and acrosin binding protein were among the most abundant interacting proteins and did interact under all three conditions. Co-immunoprecipitation and immunoblotting indicated that cystein-rich secretory protein 2 interacted with pro-acrosin (∼53 kDa) and Aacrosin binding protein under all three conditions and additionally to acrosin (∼35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein-rich secretory protein 2 was assessed via in situ proximity ligation assays. The colocalization signal of cystein-rich secretory protein 2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of cystein-rich secretory protein 2 and acrsin binding protein colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post-acrosomal sheath region upon capacitation.
DISCUSSION AND CONCLUSION
These results suggest that CRISP2 may act as a scaffold for protein complex formation and dissociation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.
Topics: Male; Animals; Swine; Acrosin; Cysteine; Semen; Spermatozoa; Proteins; Acrosome; Sperm Capacitation; Protein Binding
PubMed: 36815564
DOI: 10.1111/andr.13413 -
Reproduction & Fertility Jan 2022To summarize the currently available phase I and II clinical trials of the effects of nonoxynol-9 (N-9) on human sperm structure and functions. (Meta-Analysis)
Meta-Analysis Review
OBJECTIVE
To summarize the currently available phase I and II clinical trials of the effects of nonoxynol-9 (N-9) on human sperm structure and functions.
METHODS
A systematic review and meta-analysis aiming to evaluate the spermicidal activity of N-9 on motility, was conducted in PubMed, EMBASE, and Cochrane databases by 10 March 2021. The counted numbers of progressive motile (PR) sperm in cervical mucus and the vanguard sperm penetration distances were analyzed. Other effects on sperm structures and physiological activities were reviewed as well.
RESULTS
In the pooled results, percentages or counted numbers of PR sperm decreased after the treatment of N-9. Vanguard sperm penetration distance was shortened in treated groups. N-9 has been confirmed to damage the structures of sperm, as well as other organelles like acrosome and mitochondria. The physiological activities such as generation of reactive oxygen species, superoxide dismutase activity, acrosin activity, and hemizona binding were all inhibited in the reviewed studies.
CONCLUSIONS
N-9 has several impacts on sperm owing to its potency in reducing sperm motility and cervical mucus penetration, as well as other functional competencies.
LAY SUMMARY
Nonoxynol-9 (N-9) has been used worldwide as a spermicide to kill sperm for more than 60 years but can cause side effects including vaginal irritation and can increase the rate of contraceptive failure. A detailed analysis of published literature aiming to evaluate the spermicidal activity of N-9 on sperm was carried out. In the pooled results, N-9 reduced the number of active sperm and the distance they traveled. It also caused damage to the structures of sperm and to the way the sperm acted and interacted with the egg. In conclusion, N-9 impacts on sperm in a number of ways that lead to sperm death and dysfunction.
Topics: Female; Humans; Male; Nonoxynol; Semen; Sperm Motility; Spermatocidal Agents; Spermatozoa
PubMed: 35350652
DOI: 10.1530/RAF-21-0024 -
Biomolecules Sep 2023The global trend of rising (male) infertility is concerning, and the unidentifiable causes in half of the cases, the so-called unknown origin male infertility (UOMI),...
The global trend of rising (male) infertility is concerning, and the unidentifiable causes in half of the cases, the so-called unknown origin male infertility (UOMI), demands a better understanding and assessment of both external/internal factors and mechanisms potentially involved. In this work, it was our aim to obtain new insight on UOMI, specifically on idiopathic (ID) and Unexplained male infertility (UMI), relying on a detailed evaluation of the male gamete, including functional, metabolic and proteomic aspects. For this purpose, 1114 semen samples, from males in couples seeking infertility treatment, were collected at the Reproductive Medicine Unit from the Centro Hospitalar e Universitário de Coimbra (CHUC), from July 2018-July 2022. Based on the couples' clinical data, seminal/hormonal analysis, and strict eligibility criteria, samples were categorized in 3 groups, control (CTRL), ID and UMI. Lifestyle factors and anxiety/depression symptoms were assessed via survey. Sperm samples were evaluated functionally, mitochondrially and using proteomics. The results of Assisted Reproduction Techniques were assessed whenever available. According to our results, ID patients presented the worst sperm functional profile, while UMI patients were similar to controls. The proteomic analysis revealed 145 differentially expressed proteins, 8 of which were specifically altered in ID and UMI samples. Acrosin (ACRO) and sperm acrosome membrane-associated protein 4 (SACA4) were downregulated in ID patients while laminin subunit beta-2 (LAMB2), mannose 6-phosphate isomerase (MPI), ATP-dependent 6-phosphofructokinase liver type (PFKAL), STAR domain-containing protein 10 (STA10), serotransferrin (TRFE) and exportin-2 (XPO2) were downregulated in UMI patients. Using random forest analysis, SACA4 and LAMB2 were identified as the sperm proteins with a higher chance of distinguishing ID and UMI patients, and their function and expression variation were in accordance with the functional results. No alterations were observed in terms of lifestyle and psychological factors among the 3 groups. These findings obtained in an experimental setting based on 3 well-defined groups of subjects, might help to validate new biomarkers for unknown origin male infertility (ID and UMI) that, in the future, can be used to improve diagnostics and treatments.
Topics: Humans; Male; Semen; Semen Analysis; Proteomics; Spermatozoa; Infertility, Male
PubMed: 37892144
DOI: 10.3390/biom13101462 -
Frontiers in Endocrinology 2023Infertility is a major disease affecting human life and health, among which male factors account for about half. Asthenoteratozoospermia accounts for the majority of...
INTRODUCTION
Infertility is a major disease affecting human life and health, among which male factors account for about half. Asthenoteratozoospermia accounts for the majority of male infertility. High-throughput sequencing techniques have identified numerous variants in genes responsible for asthenoteratozoospermia; however, its etiology still needs to be studied.
METHOD
In this study, we performed whole-exome sequencing on samples from 375 patients with asthenoteratozoospermia and identified two compound heterozygous variants, a primary ciliary dyskinesia (PCD)-associated gene, in two unrelated subjects. H&E staining, SEM were employed to analyze the varies on sperm of patients, further, TEM was employed to determine the ultrastructure defects. And westernblot and immunostaining were chose to evaluate the variation of structural protein. ICSI was applied to assist the mutational patient to achieve offspring.
RESULT
We identified two HYDIN compound heterozygous variants. Patient AY078 had novel compound heterozygous splice variants (c.5969-2A>G, c.6316+1G>A), altering the consensus splice acceptor site of . He was diagnosed with male infertility and PCD, presenting with decreased sperm progressive motility and morphological abnormalities, and bronchial dilatation in the inferior lobe. Compared to the fertile control, HYDIN levels, acrosome and centrosome markers (ACTL7A, ACROSIN, PLCζ1, and Centrin1), and flagella components (TOMM20, SEPT4, SPEF2, SPAG6, and RSPHs) were significantly reduced in -deficient patients. Using intracytoplasmic sperm injection (ICSI), the patient successfully achieved clinical pregnancy. AY079 had deleterious compound heterozygous missense variants, c.9507C>G (p. Asn3169Lys) and c.14081G>A (p. Arg4694His), presenting with infertility; however, semen samples and PCD examination were unavailable.
DISCUSSION
Our findings provide the first evidence that the loss of function causes asthenoteratozoospermia presenting with various defects in the flagella structure and the disassembly of the acrosome and neck. Additionally, ICSI could rescue this failure of insemination caused by immobile and malformed sperm induced by deficiency.
Topics: Female; Humans; Male; Pregnancy; Asthenozoospermia; East Asian People; Infertility, Male; Proteins; Semen; Spermatozoa
PubMed: 36742411
DOI: 10.3389/fendo.2023.1118841 -
Annals of Translational Medicine Sep 2022Fertilization is a prerequisite for successful human reproduction. The choice of clinical fertilization strategy is crucial and directly affects clinical outcomes. This...
BACKGROUND
Fertilization is a prerequisite for successful human reproduction. The choice of clinical fertilization strategy is crucial and directly affects clinical outcomes. This study analyzes the most appropriate assisted reproductive technology (ART) strategy based on sperm parameters.
METHODS
Semen samples were divided into six groups based on semen progressive motility (PR) and semen density (SD): HMLD (high motility-low density) (PR ≥32% and sperm density <15×10/mL, n=60), HMID (high motility-intermediate density) (PR ≥32% and 15×10/mL ≤ SD <30×10/mL, n=106), HMHD (high motility-high density) (PR ≥32% and SD ≥30×10/mL, n=1,009), LMLD (low motility-low density) (PR <32% and SD <15×10/mL, n=99), LMID (low motility-intermediate density) (PR <32% and 15×10/mL ≤ SD <30×10/mL, n=77), and LMHD (low motility-high density) (PR <32% and SD ≥30×10/mL, n=164). We analyzed hyaluronic acid binding (HAB) assay and acrosin activity, along with fertilization, embryonic development, and pregnancy outcomes, to demonstrate the correlation of sperm parameters with fertilization function.
RESULTS
In the PR <32% groups, the rate of intracytoplasmic sperm injection (ICSI) treatment decreased with increasing sperm concentration. Specifically, approximately 10% of in vitro fertilization (IVF) treatment cycles required a rescue ICSI when sperm PR was <32% accompanied by SD ≥15×10/mL and PR ≥32% accompanied by SD <30×10/mL, which was significantly higher than HMHD group, P<0.001. Sperm acrosin activity and HAB ability were significantly higher in the groups with good sperm parameters, P<0.05.
CONCLUSIONS
The findings of this study suggest, fertilization ability of sperm is closely related to sperm motility and density. In clinical practice, IVF strategies should be refined based on male sperm parameters.
PubMed: 36267712
DOI: 10.21037/atm-22-4308 -
Biological Research Oct 2022Xenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a...
BACKGROUND
Xenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a suitable donor is unclear. In this study, we combined testicular culture and xenotransplantation into an integrative model and explored whether immature testicular tissue would survive and continue to develop in this model.
METHODS
In the new integrative model group, the testes of neonatal rats on postnatal day 8 (PND 8) were cultured for 4 days ex vivo and then were transplanted under the dorsal skin of castrated nude mice. The xenografted testes were resected on the 57th day after xenotransplantation and the testes of rats in the control group were harvested on PND 69. The survival state of testicular tissue was evaluated from morphological and functional perspectives including H&E staining, immunohistochemical staining of 8-OH-dG, immunofluorescence staining, TUNEL assay, ultrastructural study, gene expression and protein analysis.
RESULTS
(a) We found that complete spermatogenesis was established in the testes in the new integrative model group. Compared with the control in the same stage, the seminiferous epithelium in some tubules was a bit thinner and there were vacuoles in part of the tubules. Immunofluorescence staining revealed some ACROSIN-positive spermatids were present in seminiferous tubule of xenografted testes. TUNEL detection showed apoptotic cells and most of them were germ cells in the new integrative model group. 8-OH-dG immunohistochemistry showed strongly positive-stained in the seminiferous epithelium after xenotransplantation in comparison with the control group; (b) Compared with the control group, the expressions of FOXA3, DAZL, GFRα1, BOLL, SYCP3, CDC25A, LDHC, CREM and MKI67 in the new integrative model group were significantly elevated (P < 0.05), indicating that the testicular tissue was in an active differentiated and proliferative state; (c) Antioxidant gene detection showed that the expression of Nrf2, Keap1, NQO1 and SOD1 in the new integrative model group was significantly higher than those in the control group (P < 0.05), and DNA methyltransferase gene detection showed that the expression of DNMT3B was significantly elevated as well (P < 0.05).
CONCLUSION
The new integrative model could maintain the viability of immature testicular tissue and sustain the long-term survival in vivo with complete spermatogenesis. However, testicular genes expression was altered, vacuolation and thin seminiferous epithelium were still apparent in this model, manifesting that oxidative damage may contribute to the testicular development lesion and it needs further study in order to optimize this model.
Topics: 8-Hydroxy-2'-Deoxyguanosine; Acrosin; Animals; Antioxidants; Kelch-Like ECH-Associated Protein 1; Male; Methyltransferases; Mice; Mice, Nude; NF-E2-Related Factor 2; Rats; Spermatogenesis; Superoxide Dismutase-1; Testis
PubMed: 36195947
DOI: 10.1186/s40659-022-00398-y -
Asian Biomedicine : Research, Reviews... Dec 2020
PubMed: 37551307
DOI: 10.1515/abm-2020-0031 -
Immunity, Inflammation and Disease Dec 2021Cancer testis (CT) antigens are attractive targets for cancer immunotherapy because of their expression restriction and immunogenicity. The acrosin binding protein...
INTRODUCTION
Cancer testis (CT) antigens are attractive targets for cancer immunotherapy because of their expression restriction and immunogenicity. The acrosin binding protein (ACRBP) is a member of CT antigens. This study aimed to evaluate ACRBP expression and immunogenicity in ovarian cancer (OC).
METHODS
The expression level of ACRBP in OC tissues, normal ovarian tissues, and cell lines was detected via quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. We determined the levels of ACRBP antigen and antibody in serum samples collected from patients with OC and healthy donors using enzyme-linked immunosorbent assays (ELISA), the level of ACRBP in cell-cultured medium was also tested.
RESULTS
ACRBP mRNA and protein expressions were upregulated in OC tissues relative to normal tissue, especially highly expressed in epithelial ovarian cancer (EOC). Moreover, ACRBP expression was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage and chemosensitivity. Serological analysis showed that anti-ACRBP antibody was detected in the sera of 16 of the 56 (28.5%) patients with OC but not in healthy donors. The area under the receiver operating characteristic curve for ACRBP antibody was 0.802 (95% confidence interval [CI]: 0.708-0.876), and the sensitivity and specificity for ACRBP antibody was 85.71% and 55.0%, respectively. Kaplan-Meier analysis revealed that the overall survival (OS) and disease-free survival (DFS) in OC patients with high ACRBP expression were significantly lower than those with low expression (p = 0.040, p = 0.021). However, ACRBP antibody level was not associated with prognosis.
CONCLUSION
ACRBP expression was upregulated in OC tissues and induced humoral immune response in patients with OC, suggesting that ACRBP is a potential prognostic biomarker and a target of tumor immunotherapy for OC.
Topics: Biomarkers, Tumor; Carcinoma, Ovarian Epithelial; Carrier Proteins; Humans; Male; Ovarian Neoplasms; Prognosis; Testis
PubMed: 34528758
DOI: 10.1002/iid3.534 -
Genes Jan 2022Alkylating agents and irradiation induce testicular damage, which results in prolonged azoospermia. Even very low doses of radiation can significantly impair testis...
Alkylating agents and irradiation induce testicular damage, which results in prolonged azoospermia. Even very low doses of radiation can significantly impair testis function. However, re-irradiation is an effective strategy for locally targeted treatments and the pain response and has seen important advances in the field of radiation oncology. At present, little is known about the relationship between the harmful effects and accumulated dose of irradiation derived from continuous low-dose radiation exposure. In this study, we examined the levels of mRNA transcripts encoding markers of 13 markers of germ cell differentiation and 28 Sertoli cell-specific products in single- and re-irradiated mice. Our results demonstrated that re-irradiation induced significantly decreased testicular weights with a significant decrease in germ cell differentiation mRNA species (, , , and ). In the 13 Sertoli cell-specific mRNA species decreased upon irradiation, six mRNA species ( and ) showed significant differences between single- and re-irradiation. At the same time, different decreases in Sertoli cell-specific mRNA species were found in single-irradiation (, , , and ) and re-irradiation (-1, and -2) mice. These results indicate that long-term aspermatogenesis may differ after single- and re-irradiated treatment.
Topics: Animals; Gene Expression Profiling; Gene Expression Regulation; Male; Mice; Mice, Inbred C57BL; RNA, Messenger; Re-Irradiation; Sertoli Cells; Spermatogenesis; Testis
PubMed: 35052491
DOI: 10.3390/genes13010151