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Scientific Reports Jul 2023An in vitro spermatogenesis method using mouse testicular tissue to produce fertile sperm was established more than a decade ago. Although this culture method has...
An in vitro spermatogenesis method using mouse testicular tissue to produce fertile sperm was established more than a decade ago. Although this culture method has generally not been effective in other animal species, we recently succeeded in improving the culture condition to induce spermatogenesis of rats up to the round spermatid stage. In the present study, we introduced acrosin-EGFP transgenic rats in order to clearly monitor the production of haploid cells during spermatogenesis in vitro. In addition, a metabolomic analysis of the culture media during cultivation revealed the metabolic dynamics of the testis tissue. By modifying the culture media based on these results, we were able to induce rat spermatogenesis repeatedly up to haploid cell production, including the formation of elongating spermatids, which was confirmed histologically and immunohistochemically. Finally, we performed a microinsemination experiment with in vitro produced spermatids, which resulted in the production of healthy and fertile offspring. This is the first demonstration of the in vitro production of functional haploid cells that yielded offspring in animals other than mice. These results are expected to provide a basis for the development of an in vitro spermatogenesis system applicable to many other mammals.
Topics: Male; Rats; Mice; Animals; Spermatids; Testis; Semen; Spermatogenesis; Rats, Transgenic; Culture Media; Mammals
PubMed: 37495678
DOI: 10.1038/s41598-023-39304-1 -
Reproductive Medicine and Biology Mar 2005The evaluation of different functional sperm parameters has become a tool in andrological diagnosis. These assays determine the sperm's capability to fertilize an... (Review)
Review
The evaluation of different functional sperm parameters has become a tool in andrological diagnosis. These assays determine the sperm's capability to fertilize an oocyte. It also appears that sperm functions and semen parameters are interrelated and interdependent. Therefore, the question arose whether a given laboratory test or a battery of tests can predict the outcome in fertilization (IVF). One-hundred and sixty-one patients who underwent an IVF treatment were selected from a database of 4178 patients who had been examined for male infertility 3 months before or after IVF. Sperm concentration, motility, acrosin activity, acrosome reaction, sperm morphology, maternal age, number of transferred embryos, embryo score, fertilization rate and pregnancy rate were determined. In addition, logistic regression models to describe fertilization rate and pregnancy were developed. All the parameters in the models were dichotomized and intra- and interindividual variability of the parameters were assessed. Although the sperm parameters showed good correlations with IVF when correlated separately, the only essential parameter in the multivariate model was morphology. The enormous intra- and interindividual variability of the values was striking. In conclusion, our data indicate that the andrological status at the end of the respective treatment does not necessarily represent the status at the time of IVF. Despite a relatively low correlation coefficient in the logistic regression model, it appears that among the parameters tested, the most reliable parameter to predict fertilization is normal sperm morphology. (Reprod Med Biol 2005; 4: 7-30).
PubMed: 29699207
DOI: 10.1111/j.1447-0578.2005.00087.x -
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 -
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 -
Fertility and Sterility Apr 2009To detect the presence of antibodies to the proacrosin/acrosin system and to evaluate their effect on the sperm acrosomal protein activities in women consulting for...
Acrosin antibodies and infertility. I. Detection of antibodies towards proacrosin/acrosin in women consulting for infertility and evaluation of their effects upon the sperm protease activities.
OBJECTIVE
To detect the presence of antibodies to the proacrosin/acrosin system and to evaluate their effect on the sperm acrosomal protein activities in women consulting for infertility.
DESIGN
Retrospective study.
SETTING
Basic research laboratory.
PATIENT(S)
Recombinant proteins derived from human proacrosin (Rec-40, Rec-30, Rec-20, Rec-10) and recombinant human zona pellucida (ZP) glycoprotein A( *) (rec-hZPA).
INTERVENTION(S)
Development of an ELISA-Acro to test for antiacrosin antibodies using Rec-40 and truncated acrosin proteins as antigens.
MAIN OUTCOME MEASURE(S)
Evaluation of: 1) the presence of antiacrosin antibodies; 2) the protein regions recognized by the antibodies; 3) the relationship between antiacrosin antibodies and surface antisperm antibodies (ASA) identified by the immunobead binding test (IBT); and 4) the effect of antiacrosin antibodies upon proacrosin/acrosin binding activity to ZPA and acrosin amidase activity.
RESULT(S)
Antiacrosin antibodies were detected in sera from 34 of 179 women (19%). Detection of ASA by the IBT resulted in a similar incidence (36 of 179, 20%), although only six of them showed correspondence between both assays; five of these six sera were IBT-positive IgGs to the sperm head. Antiacrosin antibodies directed toward different protein regions inhibited proacrosin binding activity to rec-hZPA as well as its activation and acrosin amidase activity in protein sperm extracts.
CONCLUSION(S)
Antiacrosin antibodies are present in sera of women consulting for infertility in both IBT-positive and IBT-negative samples, and they affect proacrosin/acrosin activities.
Topics: Acrosin; Adult; Autoantibodies; Egg Proteins; Enzyme Activation; Enzyme Precursors; Female; Humans; Infertility, Female; Male; Membrane Glycoproteins; Peptide Hydrolases; Protein Binding; Receptors, Cell Surface; Retrospective Studies; Spermatozoa; Young Adult; Zona Pellucida Glycoproteins
PubMed: 18439585
DOI: 10.1016/j.fertnstert.2007.12.072 -
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 -
Journal of Clinical Medicine Jan 2023Different cell culture conditions and techniques have been used to mature spermatogenic cells to increase the success of in vitro fertilization. Sertoli cells (SCs) are...
Co-Culture of Cryopreserved Healthy Sertoli Cells with Testicular Tissue of Non-Obstructive Azoospermia (NOA) Patients in Culture Media Containing Follicle-Stimulating Hormone (FSH)/Testosterone Has No Advantage in Germ Cell Maturation.
Different cell culture conditions and techniques have been used to mature spermatogenic cells to increase the success of in vitro fertilization. Sertoli cells (SCs) are essential in maintaining spermatogenesis and FSH stimulation exerts its effect through direct or indirect actions on SCs. The effectiveness of FSH and testosterone added to the co-culture has been demonstrated in other studies to provide microenvironment conditions of the testicular niche and to contribute to the maturation and meiotic progression of spermatogonial stem cells (SSCs). In the present study, we investigated whether co-culture of healthy SCs with the patient's testicular tissue in the medium supplemented with FSH/testosterone provides an advantage in the differentiation and maturation of germ cells in NOA cases (N = 34). In men with obstructive azoospermia (N = 12), healthy SCs from testicular biopsies were identified and purified, then cryopreserved. The characterization of healthy SCs was done by flow cytometry (FC) and immunohistochemistry using antibodies specific for GATA4 and vimentin. FITC-conjugated annexin V/PI staining and the MTT assay were performed to compare the viability and proliferation of SCs before and after freezing. In annexin V staining, no difference was found in percentages of live and apoptotic SCs, and MTT showed that cryopreservation did not inhibit SC proliferation compared to the pre-freezing state. Then, tissue samples from NOA patients were processed in two separate environments containing FSH/testosterone and FSH/testosterone plus co-culture with thawed healthy SCs for 7 days. FC was used to measure 7th-day levels of specific markers expressed in spermatogonia (VASA), meiotic cells (CREM), and post-meiotic cells (protamine-2 and acrosin). VASA and acrosin basal levels were found to be lower in infertile patients compared to the OA group (8.2% vs. 30.6% and 12.8% vs. 30.5%, respectively; < 0.05). Compared to pre-treatment measurements, on the 7th day in the FSH/testosterone environment, CREM levels increased by 58.8% and acrosin levels increased by 195.5% ( < 0.05). Similarly, in medium co-culture with healthy SCs, by day 7, CREM and acrosin levels increased to 92.2% and 204.8%, respectively ( < 0.05). Although VASA and protamine levels increased in both groups, they did not reach a significant level. No significant difference was found between the day 7 increase rates of CREM, VASA, acrosin and protamine-2 in either FSH/testosterone-containing medium or in medium additionally co-cultured with healthy SCs (58.8% vs. 92.2%, 120.6% vs. 79.4%, 195.5% vs. 204.8%, and 232.3% vs. 198.4%, respectively; > 0.05). Our results suggest that the presence of the patient's own SCs for maturation of germ cells in the culture medium supplemented with FSH and testosterone is sufficient, and co-culture with healthy SCs does not have an additional advantage. In addition, the freezing-thawing process would not impair the viability and proliferation of SCs.
PubMed: 36769720
DOI: 10.3390/jcm12031073 -
Fertility and Sterility Jun 1992To compare biochemically active with immunoreactive sperm acrosin in fertile and infertile men.
OBJECTIVE
To compare biochemically active with immunoreactive sperm acrosin in fertile and infertile men.
SETTING
This study was conducted in a tertiary care center, the Andrology Clinic, Department of Internal Medicine, University of L'Aquila.
PATIENTS
We evaluated the males in 40 infertile couples with no recognized cause of female infertility and 20 fertile men.
INTERVENTIONS
Ejaculates were collected under standardized conditions of abstinence.
MAIN OUTCOME MEASURES
Total sperm acrosin activity was measured on a spectrophotometer in washed sperm stored at -80 degrees C for 1 to 6 days. The percent of spermatozoa immunostained by an antiserum against proacrosin/acrosin by indirect immunofluorescence (IFL) was determined on methanol fixed sperm smears.
RESULTS
Biochemically active acrosin was correlated to immunoreactive acrosin (P = 0.0028), and both were inversely correlated to the percent of spermatozoa with an abnormal head (P = 0.00024 for acrosin activity and P = 0.0013 for IFL). Biochemically active and immunoreactive acrosin were lower in infertile compared with fertile men (P = 0.0012 and P = 0.0009, respectively). Sixty-eight percent of ejaculates with an acrosin activity lower than the limit value observed in fertile men showed a normal sperm morphology and a normal immunoreactivity for acrosin.
CONCLUSIONS
A low sperm acrosin activity in teratospermic ejaculates is because of a lack or a defect of the immunogenic and functional domains of the protein. A low sperm acrosin in infertile men with normal semen parameters results from a possible functional defect of the enzyme that is immunohistochemically detected in spermatozoa.
Topics: Acrosin; Enzyme Precursors; Humans; Infertility, Male; Male; Microscopy, Fluorescence; Reference Values; Semen; Spermatozoa
PubMed: 1601156
DOI: 10.1016/s0015-0282(16)55093-6 -
Fertility and Sterility Sep 1992To review recent studies on the development of new tests of human sperm function and evaluation of which sperm characteristics are most important for fertilization in... (Review)
Review
OBJECTIVE
To review recent studies on the development of new tests of human sperm function and evaluation of which sperm characteristics are most important for fertilization in vitro by logistic regression analysis.
STUDY SELECTION
Recent studies on the relationship between putative and new tests of human sperm function and fertility in vitro or in vivo are discussed in this review. Some physiological and technical aspects are included.
MAIN OUTCOME MEASURES
Fertilization rates in vitro and sperm tests including standard semen analysis, improved morphology assessment, objective assessment of sperm motility and movement characteristics, nuclear maturity, hypo-osmotic swelling, the acrosome and the acrosome reaction, acrosin activity, human sperm-hamster oocyte penetration assay, and sperm-zona pellucida (ZP) and sperm-oolemma binding.
RESULTS
The percentages of sperm with normal morphology and a normal intact acrosome, mean linearity, and the number of sperm binding to the ZP were highly significant related to fertilization rates in vitro. Other sperm tests evaluated usually provided no additional information about fertilization rates. The human ZP is highly selective for binding of morphologically normal sperm. Acrosome-reacted human sperm have little or no ability to bind to the ZP.
CONCLUSION
Results of in vitro fertilization can be used to evaluate tests of human sperm function. Logistic regression analysis is a powerful method for determining which groups of sperm characteristics are independently related to fertilization rates. Normal morphology, linearity, acrosome status, and sperm-ZP binding are the most important characteristics for fertilization in vitro.
Topics: Acrosome; Animals; Cricetinae; Female; Fertilization in Vitro; Humans; Male; Sperm Motility; Sperm-Ovum Interactions; Spermatozoa
PubMed: 1521638
DOI: 10.1016/s0015-0282(16)55247-9 -
Animals : An Open Access Journal From... Apr 2022The spermatogenesis of crustaceans includes nuclear deformation and acrosome formation. The mechanism of acrosome formation is one focus of reproductive biology. In this...
The spermatogenesis of crustaceans includes nuclear deformation and acrosome formation. The mechanism of acrosome formation is one focus of reproductive biology. In this study, was selected as the research object to explore the mechanism of acrosome formation. The acrosome contains a large number of acrosomal enzymes for the hydrolysis of the egg envelope. How these acrosomal enzymes are transported to the acrosomal site after synthesis is the key scientific question of this study. The acroframosome (AFS) structure of caridean sperm has been reported. We hypothesized that acrosomal enzymes may be transported along the AFS framework to the acrosome by motor proteins. To study this hypothesis, we obtained the full-length cDNA sequences of and from the testis of . The and mRNA expression levels were highest in testis. We detected the distribution of Mr-KIFC1 and its colocalization with Mr-Acrosin during spermatogenesis by immunofluorescence. The colocalization of Mr-KIFC1 and microtubule indicated that Mr-KIFC1 may participate in sperm acrosome formation and nucleus maturation. The colocalization of Mr-KIFC1 and Mr-Acrosin indicated that Mr-KIFC1 may be involved in Acrosin transport during spermiogenesis of . These results suggest that Mr-KIFC1 may be involved in acrosomal enzymes transport during spermiogenesis of .
PubMed: 35454238
DOI: 10.3390/ani12080991