Abstract
Background
The epididymis is crucial for post-testicular sperm development which is termed sperm maturation. During this process, fertilizing ability is acquired through the epididymis-sperm communication via exchange of protein and small non-coding RNAs (sncRNAs). More importantly, epididymal-derived exosomes secreted by the epididymal epithelial cells transfer sncRNAs into maturing sperm. These sncRNAs could mediate intergenerational inheritance which further influences the health of their offspring. Recently, the linkage and mechanism involved in regulating sperm function and sncRNAs during epididymal sperm maturation are increasingly gaining more and more attention.
Methods
An epididymal-specific ribonuclease T2 (RNase T2) knock-in (KI) mouse model was constructed to investigate its role in develo** sperm fertilizing capability. The sperm parameters of RNase T2 KI males were evaluated and the metabolic phenotypes of their offspring were characterized. Pandora sequencing technology profiled and sequenced the sperm sncRNA expression pattern to determine the effect of epididymal RNase T2 on the expression levels of sperm sncRNAs. Furthermore, the expression levels of RNase T2 in the epididymal epithelial cells in response to environmental stress were confirmed both in vitro and in vivo.
Results
Overexpression of RNase T2 caused severe subfertility associated with astheno-teratozoospermia in mice caput epididymis, and furthermore contributed to the acquired metabolic disorders in the offspring, including hyperglycemia, hyperlipidemia, and hyperinsulinemia. Pandora sequencing showed altered profiles of sncRNAs especially rRNA-derived small RNAs (rsRNAs) and tRNA-derived small RNAs (tsRNAs) in RNase T2 KI sperm compared to control sperm. Moreover, environmental stress upregulated RNase T2 in the caput epididymis.
Conclusions
The importance was demonstrated of epididymal RNase T2 in inducing sperm maturation and intergenerational inheritance. Overexpressed RNase T2 in the caput epididymis leads to astheno-teratozoospermia and metabolic disorder in the offspring.
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Background
Paternal exposure to environmental challenges plays a critical role in maintaining the offspring’s future health, especially against those causing metabolic diseases [1, 2]. Small non-coding RNAs (sncRNAs) serve as vectors mediating paternal intergenerational inheritance. Epididymal-derived exosomes often transfer sncRNAs to spermatozoa during their post-testicular maturation process in the epididymis [3]. During the epididymal transit, sperm receive additional payloads of proteins and sncRNAs from the epididymal lumen through the epididymosomes delivery process [4, 5]. Epididymosomes are a type of exosome-like extracellular vesicle generated from the epididymal epithelial cells and are able to interact with the sperm by transferring proteins, lipids, and sncRNAs into the sperm. This process accounts for the association that exists between the epididymal epithelial cells and maturing sperm [6].
Sperm-exosome interaction is essential for sperm maturation and intergenerational inheritance of chronic disease. On the one hand, alteration of the sperm proteome during epididymal maturation is related to exosome-mediated transfer of proteins, and this process is indispensable for the acquisition of sperm motility and fertility [7]. On the other hand, the remodeling of the sncRNA profiles in maturing sperm, especially tRNA-derived small RNAs (tsRNAs, also termed tRNA fragment), depends on the exosome-mediated trafficking of sncRNAs from the epididymal epithelial cells. The sperm sncRNAs are also delivered to the embryo by fertilization and subsequently have an impact on the health of the offspring during adulthood. These outcomes show that at least some of these epididymal-acquired sncRNAs have a crucial role in intergenerational or transgenerational inheritance [1, 8]. Some studies showed that changes in protein and sncRNA expression patterns always accompanied poor sperm quality in humans [3, 9]. This association suggests that these potential characteristics can be regarded as biomarkers for assessment of male fertility, although the precise underlying mechanism of such a correlation remains unknown. More importantly, it is reported that environmental exposure, lifestyle, and pathological status could induce the changes in the sncRNAs and functional protein expression patterns in exosomes [10,11,12]. Such changes in exosomes might impair sperm maturation through crosstalk between the epididymal epithelium and sperm. As a result, they can impair normal sperm maturation, induce poor sperm quality and even intergenerational inheritance can result in ill-health of the offspring [13,14,15]. Thus, environmental stress not only can lead to poor sperm quality but also epigenetic modification induced by changes in sncRNAs can contribute to changes in intergenerational inheritance that underlie chronic disease.
Additionally, RNase T2 (also termed RNASET2 in humans), a sole member of the Rh/T2/S family of ribonucleases in humans, also exists widely in eukaryotes [16]. As a ribonuclease, RNase T2 has a broad range of biological roles, including scavenging of exogenous RNA, degradation of self-RNA, serving as extra- or intracellular cytotoxins, biogenesis of ribosomes, and immune regulation [17,18,19]. In particular, human RNASET2 is gaining much attention for its key role in cancer and inflammation. For instance, human RNASET2 possesses antitumorigenic activity through regulating macrophage polarization in the tumor microenvironment [20] and its inhibition of actin binding activity which suppresses tumor invasion and malignancy [21, 22]. On the other hand, the expression and secretion of RNase T2 can be largely induced by environmental stressors, such as inflammation or oxidative stress [Histological analysis Testes and caput epididymis fixed in Bouins’ solution overnight were embedded in paraffin and sliced into 5-μm-thick sections, followed by de-paraffinization and rehydration according to standard procedures [31]. Sections of testicular and epididymal tissue were then stained with hematoxylin and eosin (HE) and observed under a microscope (Olympus BX53, Olympus, Tokyo, Japan). The sera were isolated and analyzed as previous report [32]. Testosterone (R&D Systems, USA), anti-sperm antibodies (MLbio, Shanghai, China), and inflammatory cytokines including IL-1β, IL-6, and TNF-α (MLbio, Shanghai, China) in sera were detected, respectively, by using the immunoassay kits according to the manufacturer’s protocol. Individually housed sexually mature WT, control, or RNase T2 KI male mice (10 weeks old) were cohabited with two virgin female mice (10 weeks old) for 7 days and then separated from each other. During cohabitation, the vaginal plugs of the female mice were examined daily as evidence of mating. Twenty days after the last day of cohabitation, the number of pups produced by each mated female mouse was counted and the pregnancy rate and number of pups were analyzed [33]. The caudal epididymis was dissected and then placed in pre-warmed (37 °C) Tyrode buffer (Sigma-Aldrich, Irvine, CA, USA) to disperse the sperm. After 15 min, sperm suspensions were collected and analyzed for concentration, motility, and progressive motility by computer-assisted sperm analysis (CASA) (Hamilton-Thorn Research). For the analysis of teratozoospermia, sperm smears were fixed and stained by the Diff-Quick method (Yeasen Biotech.). In addition, sperm acrosome reaction assessed by FITC conjugated PNA (Sigma-Aldrich) staining as described [33]. Mature sperm collected from the caudal epididymis in a Percoll gradient. Then, sperm suspensions from the caudal epididymis (nearly 5 × 106/ml) were incubated with Fluo-4AM (Invitrogen, Frederick, MD, USA), merocyanine 540 (M540, Sigma-Aldrich), or tetramethyl rhodamine (TMRM, Invitrogen) for 30 min. After washing with PBS, the fluorescence signals of Fluo-4AM for calcium, M540 for sperm capacitation and TMRM for mitochondrial membrane potential were detected using flow cytometry (Becton Dickinson, Beckman Coulter, Brea, CA, USA). The Cell Quest software analyzed the emission originating from at least 30,000 events (Beckman Coulter), and three experiments were repeated for each sperm sample. Female mice were superovulated by intraperitoneal injection of 5 IU of pregnant mare’s serum gonadotropin (PROSPEC, Rehovot, Israel), and 48 h later injected with 5 IU of human chorionic gonadotropin (hCG) (Li Zhu drug plant, Zhuhai, China). Cumulus-enclosed oocyte complexes were collected 15 h after hCG administration and cultured in pre-warmed HTF media (Sigma-Aldrich). Male mice at 10-week-old were used as donor of sperm. Mature sperm were collected from the cauda epididymis and capacitated in the c-TYH medium (Sigma-Aldrich) for 30 min. For IVF experiment, 1–2 million motile sperm per milliliter were added to each oocyte complex and allowed to fertilize within 4 h. Then, the embryos were washed and cultured in HTF media overnight under oil and the numbers of 2-cell stage embryos were determined 24 h after fertilization. All fertilization embryo culture steps were carried out in a 5% CO2 atmosphere at 37 °C. For ICSI experiment, the sperm head was separated from the tail by ultrasonication, and only the sperm head was injected into the oocyte. After injection, the injected oocytes were transferred into KSOM medium (Sigma-Aldrich) at 37 °C with 5% CO2 for subsequent development. The numbers of 2-cell and 4-cell stage embryos were determined 1.5 days and 2.5 days respectively after fertilization. Detection of RNase T2 on sperm was performed by indirect immunofluorescence staining in combination with flow cytometry as described [34]. Sperm samples were labeled with mouse anti-RNase T2 monoclonal antibody (1:200 dilution, Santa Cruz Biotechnology, Texas, USA), followed by CF488-conjugated donkey anti-mouse IgG (1:400 dilution, Biotium, Hayward, CA, USA). Flow cytometry measured the fluorescence signal of sperm (Becton Dickinson, Beckman Coulter). The Cell Quest software analyzed the emission originating from at least 30,000 events (Beckman Coulter). GTT was performed as described [1]. The mice were fasted for 16 h overnight and then tested for glucose tolerance. After measuring fasting blood glucose levels, the animals received an intraperitoneal bolus of 2 g glucose per kilogram of body weight. Blood glucose concentrations at 0, 15, 30, 60, and 120 min were immediately measured with a blood glucose meter (Roche, Mannheim, Germany). The blood samples were taken from tail end of mouse. Meanwhile, serum samples were collected from the tail blood at the same times and the concentrations of blood insulin were detected by Insulin ELISA Kit (CRYSTAL CHEM INC, IL, USA). ITT was performed as described [1]. The mice were fasted for 4 h and then tested for insulin tolerance tests. After measuring fasting blood glucose levels, the animals received an intraperitoneal bolus of 0.75 IU insulin (Biosharp, Hefei, China) per kilogram of body weight. Blood glucose concentration was immediately measured at 0, 15, 30, 60, 90, and 120 min with a blood glucose meter (Roche). The blood samples were taken from tail end of mouse. The offspring of either RNase T2 KI males or control males were measured for serum biochemical indicators, including insulin, C-peptide, glucagon, leptin, adiponectin, total cholesterol (T-CHO), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglyceride (TG), and non-esterified fatty acid (NEFA). The level of insulin, C-peptide, glucagon, leptin, and adiponectin in serum were detected by relevant ELISA kits (CRYSTAL CHEM INC). The level of T-CHO, LDL-C, HDL-C, and TG were detected by relevant assay kits (Jiancheng Bioengineering Institute, Nan**g, China). The level of NEFA was detected by LabAssay™ NEFA kit (Wako, Osaka, Japan). The livers (middle lobe) of male F1 offspring at 10-week-old or 20-week-old were collected and lysed with TRIzol reagent (Invitrogen) on ice to extract RNA. RNA sequencing of mRNA in liver samples were analyzed by Shanghai Yingbai Biotechnology Co., Ltd. The mRNA levels of control-F1 group and RNase T2 KI-F1 group were compared by transcriptome sequencing. The cDNA library construction and sequencing were performed using the Illumina standard operating pipeline, and the detailed RNA-seq data were analyzed as described [35]. The DESeq algorithm was used for differential gene screening. The differentially expressed genes (DEGs) were distinguished by a false discovery rate value < 0.05. In order to annotate gene functions, DEGs were compared with GO databases. The raw transcriptome datasets have been uploaded and can be accessed in the NCBI Sequence Read Archive database (PRJNA893425, PRJNA835421). The tissues or cells were homogenized in TRIzol reagent (Invitrogen) on ice and the total RNA was extracted according to the manufacturer’s protocol. The RNA concentration and purification were detected by the NanoDrop 2000 spectrophotometer (Fisher Scientific, IL, USA). cDNA was synthesized according to PrimeScript RT kit (Takara, Dalian, China), and total cDNA was amplified with TB Green Premix Ex Taq (Takara) by 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Sequences of primers used for reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR) analysis are listed in Additional file 1: Table S1. For sperm RNA extraction, isolation of mature sperm was performed as described [1]. In short, sperm were released from the cauda epididymis into 5 ml of phosphate buffered saline (PBS) and filtered through a 40-μm cell strainer to remove tissue debris. Then, the sperm samples were treated with somatic cell lysis buffer (0.1% SDS, 0.5% TritonX-100 in DEPC H2O) and lysed in TRIzol reagent. Small RNA sequencing was performed by Shanghai Yingbai Biotechnology (Shanghai, China) to compare the small RNAs difference between RNase T2 KI sperm and control sperm. The sncRNAs were sequenced using Panoramic RNA Display by Overcoming RNA modification Aborted sequencing technology [36]. The sncRNAs in the 15–50-nt region were resolved by performing enzymatic processing to convert the 3' phosphate or 2',3' cyclic phosphate to 3'-OH and the 5 '-OH to 5'-phosphate; and removal of specific RNA methylation modifications (m1A, m1G, m3C, m22G) solved the problem that these modifications prevented the passage of reverse transcriptase. The sequencing results were presented by the small RNA annotation software SPORTS1.1(Reno, NV, USA) [37], which uncovers and annotates the original small RNAs with these modifications in the sequencing results and analyzes them together with other small RNA. Reads were mapped to the following individual sncRNAs databases sequentially: (1) the miRNA database miRBase 21 [38]; (2) the genomic tRNA database GtRNAdb [39]; (3) the rRNA databases assembled from the National Center for Biotechnology Information nucleotide and gene database; (4) the piRNA databases, including piRBase [40]. The sncRNAs species with q-value < 10% and FC > 2 were deemed differentially expressed. The raw transcriptome datasets have been uploaded and can be accessed in NCBI Sequence Read Archive database (PRJNA916976). For the primary culture of epididymal epithelial cells, the caput epididymis was isolated from the 3-week-old male mice as described [34]. In brief, the tissue fragments were dispersed by type IV collagenase (2 mg/mL) / DNase I (0.5 mg/mL) and an additional digestion by accutase cell dissociation reagent (Innovative Cell Technologies San Diego, USA). The fluid was centrifuged at 1000 × g for 5 min to remove enzymes. Finally, the epithelial cells were purified by differential adhesion and cultured in the incubator at 34 °C with 5% CO2. The purity of primary epididymal epithelial cells was routinely analyzed by immunofluorescent staining with cytokeratin 8 (a marker for epithelial cells) and vimentin (a marker for fibroblasts as negative control). For stress challenge, primary epididymal epithelial cells were treated with 2 μM H2O2 (Macklin) or 10 μg/mL of lipopolysaccharide (LPS) (Escherichia coli, O111:B4, Sigma-Aldrich) respectively for 48 h, to induce oxidative stress or inflammatory damage. The inflammation model was constructed as described [26]. Since RNase T2 activation and secretion were also induced by inflammation or exposure to oxidative stress [23], mammal RNase T2 appears to be a good candidate for future in vivo studies in order to decipher how tsRNAs are generated in mammalian cells. Of note, RNase T2 is characterized as a stress responder [23, 25, 76]. The expression and secretion of RNase T2 can be induced by tissue injury or oxidative stress. Exposure to oxidative stress induces increases in function of Rny1p which is the Rh/T2/S family homolog decreases yeast viability [25]. In mammalian cells, the expression of RNase T2 is upregulated in response to hydrogen peroxide, ultraviolet irradiation, and inflammatory stimuli and therefore lead to oxidative stress-induced apoptosis [23]. Herein, our results showed that RNase T2 was significantly upregulated in caput epididymis, EEC, and epididymal-derived exosomes in response to inflammation and oxidative stress. Thus, the harmful effects of environmental stress on sperm maturation, intergenerational inheritance, and metabolic disorder may at least partially be mediated by upregulation of epididymal RNase T2.Mice serum analyses and inflammatory cytokines analyses by ELISA
Fertility evaluation
Sperm parameters analyses
Sperm Ca2+, capacitation, and mitochondrial membrane potential measurements
In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI)
Flow cytometry assay for RNase T2 in sperm
Glucose tolerance tests (GTT) in vivo
Insulin tolerance tests (ITT) in vivo
Metabolic index analysis
Liver transcriptome analysis
RNA extraction, reverse transcription, and quantitative PCR analysis
Small RNA sequencing of sperm
The isolation, culture, and treatment of primary epididymal epithelial cells (EEC)
Induce of inflammation in mice
Conclusions
In this study, our data identify the role of RNase T2 in sperm maturation and intergenerational inheritance. We demonstrated that overexpression of RNase T2 in caput epididymis caused astheno-teratozoospermia and altered tsRNA and rsRNA profiles in sperm. RNase T2 KI-F1 showed glucose and lipid metabolism disorders. Further exploration of these mechanisms will lead to a deeper understanding of the association between sperm quality and dynamic behavior of sperm sncRNAs.
Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. The transcriptome sequencing data and small RNA-seq data were deposited in the NCBI Sequence Read Archive database under the BioProject accession number PRJNA893425, PRJNA835421 (transcriptome sequencing of liver tissue) and PRJNA916976 (small RNA-seq of sperm).
Abbreviations
- ANG:
-
Angiogenin
- Angptl4:
-
Angiopoetin-like 4
- CASA:
-
Computer-assisted sperm analysis
- DEGs:
-
Differentially expressed genes
- EEC:
-
Epididymal epithelial cells
- F1:
-
First filial generation
- GO:
-
Gene Ontology
- GTT:
-
Glucose tolerance tests
- hCG:
-
Human chorionic gonadotropin
- HDL:
-
High-density lipoprotein
- HE:
-
Hematoxylin and eosin
- HL:
-
Large head
- HLA:
-
Large amorphous head
- HLP:
-
Large pyriform head
- HLR:
-
Large round head
- HS:
-
Small head
- HSA:
-
Small amorphous head
- HST:
-
Small tapered head
- HSV:
-
Small vacuolated head
- ICSI:
-
Intracytoplasmic sperm injection
- IF:
-
Immunofluorescence
- Igfbp1:
-
Insulin-like growth factor binding protein 1
- Irs2:
-
Insulin receptor substrate 2
- ITT:
-
Insulin tolerance tests
- IVF:
-
In vitro fertilization
- KI:
-
Knock-in
- LDL:
-
Low-density lipoprotein
- LPS:
-
Lipopolysaccharide
- MC540:
-
Merocyanine 540
- miRNAs:
-
Micro-RNAs
- NEFA:
-
Non-esterified fatty acid
- Plin4:
-
Perilipin-4
- RNase T2:
-
Ribonuclease T2
- RNA-seq:
-
RNA sequencing
- rsRNAs:
-
RRNA-derived small RNAs
- RT-qPCR:
-
Real-time quantitative polymerase chain reaction
- sncRNAs:
-
Small non-coding RNAs
- T-CHO:
-
Total cholesterol
- TG:
-
Triglyceride
- TMRM:
-
Tetramethyl rhodamine methyl ester perchlorate
- tsRNAs:
-
TRNA-derived small RNAs
- WHO:
-
World Health Organization
- WT:
-
Wild type
- Zbtb16:
-
Zinc finger and BTB domain containing 16
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Acknowledgements
We appreciate the support provided by Prof. **ngxu Huang (ShanghaiTech University) for his providing Lcn5-cre mice. The authors thank Ms. Yanqin Hu (Shanghai Key Laboratory for Reproductive Medicine) for her technical assistance in histological analysis and Dr. Yong Fan (Shanghai Ninth Peoples’ Hospital) for providing the semen samples. The authors also thank Core Facility of Basic Medical Sciences (Shanghai Jiao Tong University School of Medicine) for the technical assistance in flow cytometry assay and electron microscopy analysis. The authors are very appreciative of the support provided by Prof Peter Reinach for his extensive and detailed support in improving the manuscript writing style.
Funding
This research project was supported by grants from the National Natural Science Foundation of China (No. 82171595, No. 82071694, No. 81971437, and No. 81701503) and Science and Technology Commission of Shanghai Municipality (No. 201409005800, No. 21140904000, No. 22140904200, and No. 23ZR1436000).
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Z. M. and Y. L. performed main experiments, analyzed data, and prepared the manuscript. J. L. and N. T. performed part of molecular experiments and analyzed some data. L.F. performed sperm experiments and in vitro fertilization experiments. R. F. performed the flow cytometry analysis. Y. Q. performed histological analysis. Z. X. constructed the mouse model and plasmids. Z. D. designed the project and revised the manuscript. Y. L. designed and supervised the project and provided final approval of revised the manuscript. All authors read and approved the final manuscript.
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The animal study was approved by the Ethics Committee of Shanghai Jiao Tong University School of Medicine (Approval No. A2019-029 and A2022-048). The study involving human participants was approved by the Institutional Research Ethics Committee of the Shanghai Ninth Peoples’ Hospital, Shanghai Jiao Tong University School of Medicine (Approval No. 20161205). In accordance with the principles outlined in the Declaration of Helsinki, written informed consent was obtained from patients and healthy donors for this study.
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Supplementary Information
Additional file 1: Table S1.
Sequences of primers used for RT-qPCR analysis.
Additional file 2: Figure S1.
Human RNASET2 expression in semen and spermatozoa. Figure S2. Molecular characterization of RNase T2 KI mice. Figure S3. Comparison of sperm parameters between the sperm from control mice and RNase T2 KI mice. Figure S4. Electron microscopic analysis of sperm. Figure S5. Assisted reproductive technology in sperm of RNase T2 KI and control mice. Figure S6. Inverse correlation between RNASET2 expression and spermatozoa quality in human. Figure S7. Identification of the primary epididymal epithelial cells and the isolated exosomes.
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Ma, Z., Li, J., Fu, L. et al. Epididymal RNase T2 contributes to astheno-teratozoospermia and intergenerational metabolic disorder through epididymosome-sperm interaction. BMC Med 21, 453 (2023). https://doi.org/10.1186/s12916-023-03158-1
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DOI: https://doi.org/10.1186/s12916-023-03158-1