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Cyclic AMP elevation links luteinizing hormone receptor signaling to dephosphorylation and inactivation of the NPR2 guanylyl cyclase in preovulatory mouse follicles.
Spermatogenic failure is believed to be a major cause of male infertility. The establishment of a testis organoid model would facilitate the study of such pathological mechanisms and open the possibility of male fertility preservation. Because of the complex structures and cellular events occurring within the testis, the establishment of a compartmentalized testis organoid with a complete spermatogenic cycle remains a challenge in all species. Since the late 20th century, a great variety of scaffold-based and scaffold-free testis cell culture systems have been established to recapitulate de novo testis organogenesis and in vitro spermatogenesis. The utilization of the hydrogel scaffolds provides a 3D microenvironment for testis cell growth and development, facilitating the reconstruction of de novo testis tissue-like structures and spermatogenic differentiation. Using a combination of different strategies, including the use of various scaffolding biomaterials, the incorporation of the living cells with high self-assembling capacity, and the integration of the advanced fabrication techniques, a scaffold-based testis organoid with a compartmentalized structure that supports in vitro spermatogenesis may be achieved. This article briefly reviews the current progress in the development of scaffold-based testis organoids while focusing on the scaffolding biomaterials (hydrogels), cell sources, and scaffolding approaches. Key challenges in current organoid studies are also discussed along with recommendations for future research.
Endocrine-disrupting chemicals have become an issue of scientific and public discussion. Vinclozolin (VNZ) is a fungicide that competitively antagonizes the binding of natural androgens to their receptor, disturbing the function of tissues that are sensitive to these hormones, as is the case of the male reproductive organs. A systematic review with meta-analyses of rodent studies was conducted to answer the following question: Does exposure to VNZ affect sperm parameters and testicular/epididymal weight? The methodology was prespecified according to the Cochrane Handbook for Systematic Reviews and PRISMA recommendations. Sixteen articles met the inclusion criteria, comprising a total of 1189 animals. The risk of publication bias was assessed using the Trim and Fill adjustment, funnel plot, and Egger regression test. Heterogeneity and inconsistency across the findings were tested using the Q-statistic and I2 of Higgins, respectively. Sensitivity was also analyzed. Statistical analysis was performed on Comprehensive Meta-Analysis software (Version 2.0), using random models and weighted mean differences along with a 95% confidence interval. Sperm motility, counts, daily sperm production (evidence of publication bias), and epididymis weight were decreased in VNZ-treated animals. Exposure length and dose, as well as the time point of exposure, influenced the obtained results. Despite the moderate/high heterogeneity observed, the sensitivity analysis overall demonstrated the robustness of the findings. The quality scores of the included studies were superior to 4 in a total of 9, then classified as good. The obtained data corroborate the capability of VNZ exposure to disrupt spermatogenic output and compromise male fertility.
Summary sentence
A systematic review with meta-analysis was conducted to understand how the exposure to VNZ affects fertility-related outcomes such as sperm quality parameters and testicular/epididymal weight.
Zinc is a critical component in a number of conserved processes that regulate female germ cell growth, fertility, and pregnancy. During follicle development, a sufficient intracellular concentration of zinc in the oocyte maintains meiotic arrest at prophase I until the germ cell is ready to undergo maturation. An adequate supply of zinc is necessary for the oocyte to form a fertilization-competent egg as dietary zinc deficiency or chelation of zinc disrupts maturation and reduces the oocyte quality. Following sperm fusion to the egg to initiate the acrosomal reaction, a quick release of zinc, known as the zinc spark, induces egg activation in addition to facilitating zona pellucida hardening and reducing sperm motility to prevent polyspermy. Symmetric division, proliferation, and differentiation of the preimplantation embryo rely on zinc availability, both during the oocyte development and post-fertilization. Further, the fetal contribution to the placenta, fetal limb growth, and neural tube development are hindered in females challenged with zinc deficiency during pregnancy. In this review, we discuss the role of zinc in germ cell development, fertilization, and pregnancy with a focus on recent studies in mammalian females. We further detail the fundamental zinc-mediated reproductive processes that have only been explored in non-mammalian species and speculate on the role of zinc in similar mechanisms of female mammals. The evidence collected over the last decade highlights the necessity of zinc for normal fertility and healthy pregnancy outcomes, which suggests zinc supplementation should be considered for reproductive age women at risk of zinc deficiency.
Summary sentence
An overview of the recent discoveries on the role of zinc in the reproductive processes of mammalian females including oogenesis, folliculogenesis, ovulation, maturation, fertilization, and pre- and post-implantation development.
Colony-stimulating factor 2 (CSF2) functions in the reproductive tract to modulate the function of the preimplantation embryo. The β subunit of the CSF2 receptor (CSF2RB) is not expressed in the embryo, and signal transduction is therefore different than for myeloid cells where the receptor is composed of α (CSF2RA) and β subunits. Here, we produced embryos in which exons 5 and 6 of CSF2RA were disrupted using the CRISPR/Cas 9 system to test whether CSF2RA signaling was essential for actions of CSF2 in the bovine embryo. Wild-type and CSF2RA knockout embryos were treated with 10 ng/mL CSF2 or vehicle at day 5 of development. Blastocysts were harvested at day 8 to determine transcript abundance of 90 genes by real-time polymerase chain reaction (PCR). Responses in female blastocysts were examined separately from male blastocysts because actions of CSF2 are sex-dependent. For wild-type embryos, CSF2 altered expression of 10 genes in females and 20 in males. Only three genes were affected by CSF2 in a similar manner for both sexes. Disruption of CSF2RA prevented the effect of CSF2 on expression for 9 of 10 CSF2-regulated genes in females and 19 of 20 genes in males. The results confirm the importance of CSF2RA for regulation of gene expression by CSF2 in the blastocyst.
Summary sentence
Disruption of CSF2RA blocked most CSF2-induced changes in gene expression in the blastocyst, indicating that, despite the absence of the β subunit of the receptor, the preimplantation embryo uses CSF2RA for signal transduction.
N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Here, we show that METTL3-mediated m6A methylation sustains porcine blastocyst development via negatively modulating autophagy. We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 (a key regulator of autophagy) and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3'UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell qPCR revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Taken together, these results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.
Summary sentence
METTL3 sustains porcine blastocyst development via inhibiting autophagy.
Reproductive efficiency in livestock is a major driver of sustainable food production. The poorly understood process of ruminant conceptus elongation (a) prerequisites maternal pregnancy recognition, (b) is essential to successful pregnancy establishment, and (c) coincides with a period of significant conceptus mortality. Conceptuses at five key developmental stages between Days 8–16 were recovered and cultured in vitro for 6 h prior to conditioned media analysis by untargeted ultrahigh-performance liquid chromatography tandem mass spectroscopy. This global temporal biochemical interrogation of the ex situ bovine conceptus unearths two antithetical stage-specific metabolic phenotypes during tubular (metabolically retentive) vs. filamentous (secretory) development. Moreover, the retentive conceptus phenotype on Day 14 coincides with an established period of elevated metabolic density in the uterine fluid of heifers with high systemic progesterone—a model of accelerated conceptus elongation. These data, combined, suggest a metabolic mechanism underpinning conceptus elongation, thereby enhancing our understanding of the biochemical reciprocity of maternal–conceptus communication, prior to maternal pregnancy recognition.
Summary sentence
Temporal changes in conceptus metabolism occur in the period leading to maternal pregnancy recognition.
Differentiation of endometrial stromal cells (ESCs) into secretory decidualized cells (dESCs) is essential for embryo implantation. Adenomyosis is a common benign gynecological disease that causes infertility. However, whether adenomyosis affects decidualization of human ESCs is elusive. Primary eutopic ESCs were obtained from patients with adenomyosis (n = 9) and women with nonendometrial diseases (n = 12). We determined the capacity of decidualization of human ESCs by qRT-PCR, Edu proliferation assay, cytokine array, and ELISA assay. We found that the expression of decidualization markers (IGFBP1 and PRL) in ESCs of adenomyosis was reduced, concomitant with increased cell proliferation. Differential secretion of cytokines in dESCs, including CXCL1/2/3, IL-6, IL-8, MCP-1, VEGF-A, MIP-3α, OPN, SDF-1α, HGF, and MMP-9, was observed between adenomyosis and nonadenomyosis. Moreover, the expression of decidualization regulators (HOXA10 at both mRNA and protein levels, FOXO1, KLF5, CEBPB, and HAND2 at mRNA levels) in the eutopic endometrium of adenomyosis was lower than that of nonadenomyosis. We propose that ESCs from adenomyosis have defected ability to full decidualization, which may lead to a nonreceptive endometrium.
Summary Sentence. The endometrial stromal cells of adenomyosis exhibit an aberrant decidualization response, accompanied by changes in the secretion of cytokines and matrix metalloproteinases, which may cause an unreceptive endometrium.
Cripto encodes for a cell surface receptor whose role in embryonic development and stem cell maintenance has been studied. Cripto mRNA and protein have been detected in the human uterus at all stages of the menstrual cycle. To date, there is not much known about Cripto's role in female reproduction. As Cripto null Knockout (KO) is embryonic lethal, we created a conditional KO (cKO) mouse model in which Cripto is deleted only in the reproductive tissues using a Cre-loxP system. Pregnancy rate and number of pups per litter were evaluated as general fertility indices. We observed a significant decrease in pregnancy rate and litter size with loss of uterine Cripto indicating that Cripto cKO females are subfertile. We showed that although the preimplantation period is normal in Cripto cKO females, 20% of cKO females fail to establish pregnancy and an additional 20% of females undergo full litter loss after implantation between day 5.5 postcoitum (d5.5pc) and d8.5pc. We showed that subfertility caused by loss of uterine Cripto is due to defects in uterine decidualization, remodeling, and luminal closure and is accompanied by significant downregulation of Bmp2, Wnt4 and several components of Notch signaling pathway which all are known to be important factors in uterine remodeling and decidualization. Our study demonstrates that Cripto is expressed in the uterus during critical stages of early pregnancy and its deletion results in subfertility due to implantation failure, impaired peri-implantation uterine remodeling and impaired uterine decidualization.
Summary sentence
Cripto is involved in uterine luminal closure and stromal decidualization.
Oocytes are highly radiosensitive, so agents that prevent radiation-induced ovarian follicle destruction are important fertility preservation strategies. A previous study in rhesus macaques demonstrated that ovarian treatment with antiapoptotic agents, sphingosine-1-phosphate (S1P) and FTY720, its long-acting mimetic, preserved follicles following a single dose of 15 Gy X-ray radiation, and live offspring were obtained from FTY720-treated animals. However, it is unknown whether these antiapoptotic agents also protected the ovarian stroma from late effects of radiation, including vascular damage and fibrosis. Using ovarian histological sections from this study, we evaluated the vasculature and extracellular matrix in the following cohorts: vehicle + sham irradiation, vehicle + irradiation (OXI), S1P + irradiation (S1P), and FTY720 + irradiation (FTY720). One ovary from each animal was harvested prior to radiation whereas the contralateral ovary was harvested 10 months post-treatment. We assessed vasculature by immunohistochemistry with a PECAM1 antibody, hyaluronan by a hyaluronan binding protein assay, and collagen by picrosirius red and Masson's trichrome staining. Disorganized vessels were observed in the medulla in the OXI and S1P cohorts relative to the sham, but the vasculature in the FTY720 cohort appeared intact, which may partially explain fertoprotection. There were no differences in the hyaluronan matrix among the cohorts, but there was thickening of the tunica albuginea and fibrosis in the OXI cohort relative to the sham, which was not mitigated by either S1P or FTY720 treatment. Thus, the fertoprotective properties of S1P and FTY720 may be limited given their inability to protect the ovarian stroma against the late effects of radiation-induced fibrosis.
Summary sentence: Although the fertoprotective agents S1P and FTY720 preserve the ovarian reserve, they do not protect the ovarian stroma from radiation-induced fibrosis in the nonhuman primate, underscoring the need to prevent damage in the ovarian microenvironment.
Calcitonin gene-related peptide (CALCB), adrenomedullin (ADM), and adrenomedullin2 (ADM2) are hypotensive peptides that belong to CALCB family of peptides. Goal of this study was to identify the effect of fms-like tyrosine kinase (sFLT-1) and angiotensin2 (Ang2) on the function of these peptides in OA smooth muscle cells (OASMC) and assess the sensitivity of OA for these peptides in preeclampsia (PE) and normotensive pregnancy. Methods: Peptide function was assessed by Cyclic adenosine monophosphate (cAMP) assays and wire myograph; mRNA expression by Polymerase chain reaction (PCR) and protein-protein interaction by proximity ligation assay and co-immunoprecipitation. Findings: All three peptides increased cAMP synthesis in the order of efficacy CALCB > ADM = ADM2 and vascular endothelial growth factor (VEGF) mRNA in OASMC (P < 0.05); sFLT-1 mediated decrease in cAMP synthesis (P < 0.05) is differentially rescued by all three CALCB family peptides in OASMC (P < 0.005); sFLT-1 decreased receptor activity-modifying protein (RAMP)1 and RAMP2 mRNA expression (P < 0.05); Ang2 decreased the expression of calcitonin-receptor-like receptor and RAMP1 mRNA and desensitized CALCB and ADM2 receptors in OASMC (P < 0.05); sFLT-1 increased RAMP1and Ang2 type 1 receptor (AT1R) interaction in OASMC which is inhibited in presence of all three peptides; and all three peptides relax OA in PE with enhanced ADM2 response (P < 0.05). Conclusion: sFLT-1 and Ang2 impair OASMC mediated functional responses of CALCB family peptides which can be inhibited by respective peptide treatment. The sensitivity of OA for CALCB, ADM, and ADM2-mediated relaxation is retained in PE.
Summary sentence
sFLT-1 and Ang2 impair smooth muscle cell mediated functional responses of CALCB family peptides which can be inhibited by respective peptide treatment.
Appropriate mineralization of the fetal skeleton requires an excess of phosphate in the fetus compared to the mother. However, mechanisms for placental phosphate transport are poorly understood. This study aimed to identify phosphate regulatory pathways in ovine endometria and placentae throughout gestation. Suffolk ewes were bred with fertile rams upon visual detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n = 3–14/Day), ewes were euthanized and hysterectomized. Phosphate abundance varied across gestational days in uterine flushings, allantoic fluid, and homogenized endometria and placentae (P < 0.05). The expression of mRNAs for sodium-dependent phosphate transporters (SLC20A1 and SLC20A2) and klotho signaling mediators (FGF7, FGF21, FGF23, FGFR1–4, KL, KLB, ADAM10, and ADAM17) were quantified by qPCR. Day 17 conceptus tissue expressed SLC20A1, SLC20A2, KLB, FGF7, FGF21, FGF23, FGFR1, and FGFR2 mRNAs. Both sodium-dependent phosphate transporters and klotho signaling mediators were expressed in endometria and placentae throughout gestation. Gestational day influenced the expression of SLC20A1, ADAM10, ADAM17, FGF21, FGFR1, and FGFR3 mRNAs in both endometria and placentae (P < 0.05). Gestational day influenced endometrial expression of FGF7 (P < 0.001), and placental expression of FGF23 (P < 0.05). Immunohistochemistry confirmed that both FGF23 and KL proteins were expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile of KL-FGF signaling suggests a potential role in the establishment of pregnancy and regulation of fetal growth. This study provides a platform for further mechanistic investigation into the role for KL-FGF signaling in the regulation of phosphate transport at the ovine maternal–conceptus interface.
Summary sentence
Components of Klotho-FGF signaling and sodium-dependent phosphate transporters are present at the ovine conceptus-maternal interface, suggesting a potential role in phosphate transport throughout gestation.
Gemma Gaitskell-Phillips, Francisco E. Martín-Cano, José M. Ortiz-Rodríguez, Antonio Silva-Rodríguez, Maria C. Gil, Cristina Ortega-Ferrusola, Fernando J. Peña
The identification of stallions and or ejaculates that will provide commercially acceptable quality post-thaw before cryopreservation is of great interest, avoiding wasting time and resources freezing ejaculates that will not achieve sufficient quality to be marketed. Our hypothesis was that after bioinformatic analysis, the study of the stallion sperm proteome can provide discriminant variables able to predict the post-thaw quality of the ejaculate. At least three ejaculates from 10 different stallions were frozen following a split sample design. Half of the ejaculate was analyzed as a fresh aliquot and the other half was frozen and then analyzed as a frozen-thawed aliquot. Computer-assisted sperm analysis and flow cytometry were used to analyze sperm quality. Detailed proteomic analysis was performed on fresh and frozen and thawed aliquots, and bioinformatic analysis was used to identify discriminant variables in fresh samples able to predict the outcome of cryopreservation. Those with a fold change > 3, a P = 8.2e-04, and a q = 0.074 (equivalent to False discovery rate (FDR)) were selected, and the following proteins were identified in fresh samples as discriminant variables of good motility post-thaw: F6YTG8, K9K273, A0A3Q2I7V9, F7CE45, F6YU15, and F6SKR3. Other discriminant variables were also identified as predictors of good mitochondrial membrane potential and viability post-thaw. We concluded that proteomic approaches are a powerful tool to improve current sperm biotechnologies.
Conventional heterologous mitochondrial replacement therapy is clinically complicated by “triparental” ethical concerns and limited source of healthy donor oocytes or zygotes. Autologous mitochondrial transfer is a promising alternative in rescuing poor oocyte quality and impaired embryo developmental potential associated with mitochondrial disorders, including aging. However, the efficacy and safety of mitochondrial transfer from somatic cells remains largely controversial, and unsatisfying outcomes may be due to distinct mitochondrial state in somatic cells from that in oocytes. Here, we propose a potential strategy for improving in vitro fertilization (IVF) outcomes of aging female patients via mitochondrial transfer from induced pluripotent stem (iPS) cells. Using naturally aging mice and well-established cell lines as models, we found iPS cells and oocytes share similar mitochondrial morphology and functions, whereas the mitochondrial state in differentiated somatic cells is substantially different. By microinjection of isolated mitochondria into fertilized oocytes following IVF, our results indicate that mitochondrial transfer from iPS, but not MEF cells, can rescue the impaired developmental potential of embryos from aging female mice and obtain an enhanced implantation rate following embryo transfer. The beneficial effect may be explained by the fact that mitochondrial transfer from iPS cells not only compensates for aging-associated loss of mtDNA, but also rescues mitochondrial metabolism of subsequent preimplantation embryos. Using mitochondria from iPS cells as the donor, our study not only proposes a promising strategy for improving IVF outcomes of aging females, but also highlights the importance of synchronous mitochondrial state in supporting embryo developmental potential.
Summary Sentence: Induced pluripotent stem cells are more preferred donors for mitochondrial transfer than differentiated somatic cells, for rescuing the impaired developmental potential of IVF embryos from aging females.
The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.
Summary sentence
Activation of bovine oocytes by protein synthesis inhibitors modulate MPF by CDK1-dependent specific phosphorylation and via an MAPKs-independent pathway.
It has been suggested that many novel RNA-binding proteins (RBPs) are required for gametogenesis, but the necessity of few of these proteins has been functionally verified. Here, we identified one RBP, Rbm46, and investigated its expression pattern and role in zebrafish reproduction. We found that rbm46 is maternally provided and specifically expressed in the germ cells of gonadal tissues using in situ hybridization, reverse transcription-PCR, and quantitative real-time polymerase chain reaction (qRT-PCR). Two independent rbm46 mutant zebrafish lines were generated via the transcription activator-like effector nuclease technique. Specific disruption of rbm46 resulted in masculinization and infertility in the mutants. Although the spermatogonia appeared grossly normal in the mutants, spermatogenesis was impaired, and meiosis events were not observed. The introduction of a tp53M214K mutation could not rescue the female-to-male sex-reversal phenotype, indicating that rbm46 acts independently of the p53-dependent apoptotic pathway. RNA sequencing and qRT-PCR subsequently indicated that Rbm46 might be involved in the posttranscriptional regulation of functional genes essential for germ cell development, such as nanos3, dazl, and sycp3, during gametogenesis. Together, our results reveal for the first time the crucial role of rbm46 in regulating germ cell development in vivo through promotion of germ cell progression through meiosis prophase I.
Summary sentence
We generated zebrafish with rbm46 mutations using transcription activator-like effector nucleases to demonstrate that the disruption of rbm46 results in masculinization and infertility through impairment of germ cell progression through meiosis prophase I.
Graphical Abstract
Schematic diagram showing the proposed function of Rbm46, which is critical for germ cell progression through meiosis prophase I in zebrafish. All the mutants are infertile males with spermatogonia or premeiotic cells. Meiosis defects were observed and Rbm46 is potentially involved in the posttranscriptional regulation of functional genes during spermatogenesis, such as nanos3, dazl, sycp3, in mutants.
Sperm quality is an important indicator of male fertility, and a suitable biomarker enables the selection of high-quality spermatozoa. We previously found that L-amino acid oxidase encoded by the L-amino acid oxidase 1 (Lao1) gene exerts biological roles in the mammary gland and brain by converting specific L-amino acids into keto acids, ammonia, and hydrogen peroxide (H2O2). Here, we describe the role of Lao1 in male reproduction. Lao1-deficient (Lao1–/–) male mice generated fewer pregnant embryos and pups as well as lower ratios of fertilized oocytes and even ovulated number was not different, suggesting that male subfertility caused the smaller litters. We found that LAO1 expressed in acrosomes is associated with high malformation ratios and low viability of Lao1–/– sperm. Wild-type (WT) sperm produced more H2O2 than Lao1–/– sperm, and 10 µM H2O2 restored knockout (KO) sperm viability in vitro. In addition, the sperm ratio of induced acrosome reaction was higher in WT than in Lao1–/– sperm incubated with the calcium ionophore A23187. Moreover, LAO1 expression was abundant in bovine sperm with high fertilization ratios. We concluded that LAO1 localized in the sperm acrosome influences sperm viability and morphology as well as the acrosome reaction, and that LAO1-deficient sperm might cause male subfertility. Thus, LAO1 might serve as a novel marker for selecting high-quality spermatozoa, especially for livestock reproduction.
Summary sentence
LAO1 localized in the sperm acrosome may influence sperm viability and acrosome reaction by metabolizing L-amino acid to produce H2O2.
Atrazine (ATZ) is an extensively used herbicide and ubiquitous environmental contaminant. ATZ and its metabolite, diaminochlorotriazine (DACT), cause several cellular and functional alterations in spermatozoa. We aimed to examine the effect of ATZ/DACT on spermatozoon DNA integrity, fertilization competence, embryonic development, and transcriptome profile of in vitro-produced embryos derived from fertilization with pre-exposed sperm. Bovine spermatozoa exposed to ATZ (0.1 or 1 µM) or DACT (1 or 10 µM) during in vitro capacitation were used for in vitro fertilization of untreated oocytes. Cleavage and blastocyst-formation rates were evaluated 42 h and 7 days postfertilization, respectively. The association between DNA fragmentation and apoptosis (annexin V kit) was determined. Fertilization competence of annexin-positive (AV+) and annexin-negative (AV–) spermatozoa was examined. Microarray analysis was performed for 7-day blastocysts. Intracytoplasmic sperm injection was performed with control (AV+, AV–) and DACT (AV+, AV–) spermatozoa. Cleavage rates did not differ between groups and blastocyst formation tended to be higher for AV– vs. AV+ in both control and DACT groups, suggesting that acrosome reaction, rather than DNA fragmentation, underlies the reduced cleavage. Transcriptomic analysis revealed 139 and 230 differentially expressed genes in blastocysts derived from ATZ- and DACT-exposed spermatozoa, respectively, relative to controls. Proteomic analysis shown differential expression of proteins in ATZ- or DACT-treated spermatozoa, in particular proteins related to cellular processes and biological pathways. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. Overall, the current study reveals a deleterious carryover effect of ATZ/DACT from the spermatozoa to the developing embryo.
Summary sentence
Summary sentence: Atrazine and its metabolite, diaminochlorotriazine, induce multifactorial damage in sperm, which is carried over to the embryo's transcriptome.
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