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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 -
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 -
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 -
International Journal of Molecular... Oct 2021Leukemia and treatment of male patients with anticancer therapy (aggressive chemotherapy and/or radiotherapy) may lead to infertility or even permanent male sterility....
Leukemia and treatment of male patients with anticancer therapy (aggressive chemotherapy and/or radiotherapy) may lead to infertility or even permanent male sterility. Their mechanisms of spermatogenesis impairment and the decrease in male fertility are not yet clear. We showed that under acute myeloid leukemia (AML) conditions, alone and in combination with cytarabine (CYT), there was significant damage in the histology of seminiferous tubules, a significant increase in apoptotic cells of the seminiferous tubules, and a reduction in spermatogonial cells (SALL and PLZF) and in meiotic (CREM) and post-meiotic (ACROSIN) cells. In addition, we showed a significant impairment in sperm parameters and fertilization rates and offspring compared to control. Our results showed a significant decrease in the expression of glial cell line-derived neurotrophic factor (GDNF), macrophage colony-stimulating factor (MCSF) and stem cell factor (SCF) under AML conditions, but not under cytarabine treatment compared to control. In addition, our results showed a significant increase in the pro-inflammatory cytokine interleukin-1 (IL-1) alpha in whole testis homogenates in all treatment groups compared to the control. Increase in IL-1 beta level was shown under AML conditions. We identified for the first time the expression of GCSF receptor (GCSFR) in sperm cells. We showed that GCSF injection in combination with AML and cytarabine (AML + CYT + GCSF) extended the survival of mice for a week (from 6.5 weeks to 7.5 weeks) compared to (AML + CYT). Injection of GCSF to all treated groups (post hoc), showed a significant impact on mice testis weight, improved testis histology, decreased apoptosis and increased expression of pre-meiotic, meiotic and post- meiotic markers, improved sperm parameters, fertility capacity and number of offspring compared to the controls (without GCSF). GCSF significantly improved the spermatogonial niche expressed by increased the expression levels of testicular GDNF, SCF and MCSF growth factors in AML-treated mice and (AML + CYT)-treated mice compared to those groups without GCSF. Furthermore, GCSF decreased the expression levels of the pro-inflammatory cytokine IL-12, but increased the expression of IL-10 in the interstitial compartment compared to the relevant groups without GCSF. Our results show for the first time the capacity of post injection of GCSF into AML- and CYT-treated mice to improve the cellular and biomolecular mechanisms that lead to improve/restore spermatogenesis and male fertility. Thus, post injection of GCSF may assist in the development of future therapeutic strategies to preserve/restore male fertility in cancer patients, specifically in AML patients under chemotherapy treatments.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cells, Cultured; Disease Models, Animal; Fertility; Granulocyte Colony-Stimulating Factor; Infertility, Male; Leukemia, Myeloid, Acute; Male; Mice; Mice, Inbred C57BL; Spermatogenesis; Spermatogonia; Testis
PubMed: 34681817
DOI: 10.3390/ijms222011157 -
Asian Journal of Andrology 2015In vitro culture of spermatogonial stem cells (SSCs) has generally been performed using two-dimensional (2D) culture systems; however, such cultures have not led to the... (Review)
Review
In vitro culture of spermatogonial stem cells (SSCs) has generally been performed using two-dimensional (2D) culture systems; however, such cultures have not led to the development of complete spermatogenesis. It seems that 2D systems do not replicate optimal conditions of the seminiferous tubules (including those generated by the SSC niche) and necessary for spermatogenesis. Recently, one of our laboratories has been able to induce proliferation and differentiation of mouse testicular germ cells to meiotic and postmeiotic stages including generation of sperm in a 3D soft agar culture system (SACS) and a 3D methylcellulose culture system (MCS). It was suggested that SACS and MCS form a special 3D microenvironment that mimics germ cell niche formation in the seminiferous tubules, and thus permits mouse spermatogenesis in vitro. In this review, we (1) provide a brief overview of the differences in spermatogenesis in rodents and primates, (2) summarize data related to attempts to generate sperm in vitro, (3) report for the first time formation of colonies/clusters of cells and differentiation of meiotic (expression of CREM-1) and postmeiotic (expression of acrosin) germ cells from undifferentiated spermatogonia isolated from the testis of prepubertal rhesus monkeys and cultured in SACS and MCS, and (4) indicate research needed to optimize 3D systems for in vitro primate spermatogenesis and for possible future application to man.
Topics: Acrosin; Agar; Animals; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cyclic AMP Response Element Modulator; Macaca mulatta; Male; Methylcellulose; Mice; Spermatogenesis; Spermatogonia; Spermatozoa
PubMed: 26067870
DOI: 10.4103/1008-682X.154994 -
Scientific Reports Jul 2022Prepubertal cancer treatment leads to irreversible infertility in half of the male patients. Current in vitro spermatogenesis protocols and cryopreservation techniques...
Prepubertal cancer treatment leads to irreversible infertility in half of the male patients. Current in vitro spermatogenesis protocols and cryopreservation techniques are inadequate to expand spermatogonial stem/progenitor cells (SSPC) from testicles. Bone marrow derived mesenchymal stem cells (BM-MSC) bearing a close resemblance to Sertoli cells, improved spermatogenesis in animal models. We asked if a co-culture setup supported by syngeneic BM-MSC that contributes to the air-liquid interphase (ALI) could lead to survival, expansion and differentiation of SSPCs in vitro. We generated an ALI platform able to provide a real-time cellular paracrine contribution consisting of syngeneic BM-MSCs to neonatal C57BL/6 mice testes. We aimed to evaluate the efficacy of this culture system on SSPC pool expansion and spermatogenesis throughout a complete spermatogenic cycle by measuring the number of total germ cells (GC), the undifferentiated and differentiating spermatogonia, the spermatocytes and the spermatids. Furthermore, we evaluated the testicular cell cycle phases, the tubular and luminal areas using histochemical, immunohistochemical and flow cytometric techniques. Cultures in present of BM-MSCs displayed survival of ID4(+) spermatogonial stem cells (SSC), expansion of SALL4(+) and OCT4(+) SSPCs, VASA(+) total GCs and Ki67(+) proliferative cells at 42 days and an increased number of SCP3(+) spermatocytes and Acrosin(+) spermatids at 28 days. BM-MSCs increased the percentage of mitotic cells within the G2-M phase of the total testicular cell cycle increased for 7 days, preserved the cell viability for 42 days and induced testicular maturation by enlargement of the tubular and luminal area for 42 days in comparison to the control. The percentage of PLZF(+) SSPCs increased within the first 28 days of culture, after which the pool started to get smaller while the number of spermatocytes and spermatids increased simultaneously. Our findings established the efficacy of syngeneic BM-MSCs on the survival and expansion of the SSPC pool and differentiation of spermatogonia to round spermatids during in vitro culture of prepubertal mice testes for 42 days. This method may be helpful in providing alternative cures for male fertility by supporting in vitro differentiated spermatids that can be used for round spermatid injection (ROSI) to female oocyte in animal models. These findings can be further exploited for personalized cellular therapy strategies to cure male infertility of prepubertal cancer survivors in clinics.
Topics: Adult Germline Stem Cells; Animals; Animals, Newborn; Cell Differentiation; Female; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Spermatogenesis; Spermatogonia; Testis
PubMed: 35798781
DOI: 10.1038/s41598-022-15358-5 -
Andrology Nov 2019Previous studies suggested that sperm head shape may serve as an effective indicator of semen quality. However, there lacks research with large sample and quantitative...
BACKGROUND
Previous studies suggested that sperm head shape may serve as an effective indicator of semen quality. However, there lacks research with large sample and quantitative measurement.
OBJECTIVES
The objective of this retrospective study was to explore the association between sperm head elongation (Width/Length ratio) and routine semen parameters.
MATERIALS AND METHODS
From January 2012 to December 2017, 63 866 semen samples were collected from male subjects at 18-60 years of age. Sperm head elongation and routine semen parameters (semen volume, sperm concentration, motility, etc.) were examined with computer-assisted semen analysis (CASA) systems in order to evaluate the association between elongation and semen quality.
RESULTS
Logistic and linear regression models showed that the value of elongation is negatively correlated with sperm concentration, total sperm count, progressive motility, total motility, percentage of morphologically normal spermatozoa, and acrosin activity (all p < 0.001).
DISCUSSION
The results suggested that higher value of elongation is generally associated with higher risks of abnormality in semen quality. The importance of elongation may be explained by abnormal acrosin activity in the round-headed spermatozoa, which has been reported to cause failure of natural pregnancy.
CONCLUSIONS
This study provides a new insight into the sperm head shape, which may be used as a complementary parameter in clinical semen examination and academic research.
Topics: Acrosin; Adolescent; Adult; Humans; Infertility, Male; Male; Middle Aged; Retrospective Studies; Semen; Semen Analysis; Sperm Count; Sperm Head; Sperm Motility; Teratozoospermia; Young Adult
PubMed: 30934170
DOI: 10.1111/andr.12619 -
PloS One 2023Mouse spermatogenesis, from spermatogonial stem cell proliferation to sperm formation, can be reproduced in vitro by culturing testis tissue masses of neonatal mice....
Mouse spermatogenesis, from spermatogonial stem cell proliferation to sperm formation, can be reproduced in vitro by culturing testis tissue masses of neonatal mice. However, it remains to be determined whether this method is also applicable when testis tissues are further divided into tiny fragments, such as segments of the seminiferous tubule (ST), a minimal anatomical unit for spermatogenesis. In this study, we investigated this issue using the testis of an Acrosin-GFP/Histone H3.3-mCherry (Acr/H3) double-transgenic mouse and monitored the expression of GFP and mCherry as indicators of spermatogenic progression. Initially, we noticed that the cut and isolated stretches of ST shrunk rapidly and conglomerated. We therefore maintained the isolation of STs in two ways: segmental isolation without truncation or embedding in soft agarose. In both cases, GFP expression was observed by fluorescence microscopy. By whole-mount immunochemical staining, meiotic spermatocytes and round and elongating spermatids were identified as Sycp3-, crescent-form GFP-, and mCherry-positive cells, respectively. Although the efficiency was significantly lower than that with tissue mass culture, we clearly showed that spermatogenesis can be induced up to the elongating spermatid stage even when the STs were cut into short segments and cultured in isolation. In addition, we demonstrated that lowered oxygen tension was favorable for spermatogenesis both for meiotic progression and for producing elongating spermatids in isolated STs. Culturing isolated STs rather than tissue masses is advantageous for explicitly assessing the various environmental parameters that influence the progression of spermatogenesis.
Topics: Male; Mice; Animals; Spermatogonia; Semen; Seminiferous Tubules; Spermatogenesis; Testis; Spermatids; Mice, Transgenic
PubMed: 37023052
DOI: 10.1371/journal.pone.0283773 -
International Journal of Molecular... Apr 2024Gonadotoxic agents could impair spermatogenesis and may lead to male infertility. The present study aimed to evaluate the effect of IL-1β on the development of...
Gonadotoxic agents could impair spermatogenesis and may lead to male infertility. The present study aimed to evaluate the effect of IL-1β on the development of spermatogenesis from cells isolated from seminiferous tubules (STs) of normal and busulfan-treated immature mice in vitro. Cells were cultured in a 3D in vitro culture system for 5 weeks. We examined the development of cells from the different stages of spermatogenesis by immunofluorescence staining or qPCR analyses. Factors of Sertoli and Leydig cells were examined by qPCR analysis. We showed that busulfan (BU) treatment significantly reduced the expression of testicular IL-1β in the treated mice compared to the control group (CT). Cultures of cells from normal and busulfan-treated immature mice induced the development of pre-meiotic (Vasa), meiotic (Boule), and post-meiotic (acrosin) cells. However, the percentage of developed Boule and acrosin cells was significantly lower in cultures of busulfan-treated mice compared to normal mice. Adding IL-1β to both cultures significantly increased the percentages of Vasa, Boule, and acrosin cells compared to their controls. However, the percentage of Boule and acrosin cells was significantly lower from cultures of busulfan-treated mice that were treated with IL-1β compared to cultures treated with IL-1β from normal mice. Furthermore, addition of IL-1β to cultures from normal mice significantly increased only the expression of androgen receptor and transferrin but no other factors of Sertoli cells compared to their CT. However, the addition of IL-1β to cultures from busulfan-treated mice significantly increased only the expression of androgen-binding protein and the FSH receptor compared to their CT. Adding IL-1β to cultures of normal mice did not affect the expression of 3βHSD compared to the CT, but it significantly reduced its expression in cultures from busulfan-treated mice compared to the CT. Our findings demonstrate the development of different stages of spermatogenesis in vitro from busulfan-treated mice and that IL-1β could potentiate this development in vitro.
Topics: Animals; Busulfan; Spermatogenesis; Male; Interleukin-1beta; Mice; Sertoli Cells; Testis; Leydig Cells; Seminiferous Tubules; Cells, Cultured
PubMed: 38732137
DOI: 10.3390/ijms25094926