Sperm microRNAs confer depression susceptibility to offspring

Behavioral and gene alterations of chronic mild stress–induced F0 males of depression-like model

Stress and resilience play critical roles in the development of depression (22–24). Therefore, we first established a chronic mild stress (CMS)–induced depression-like mouse model by exposing male mice to unpredictable mild stresses daily for 5 weeks (Fig. 1A; detailed experimental design is shown in fig. S1). As expected, the CMS-induced F0 males of depression-like model (F0-Dep) showed decreased body weight gain compared to nonstressed F0 control males (F0-Ctl) (Fig. 1B). Moreover, F0-Dep displayed obvious depression-like behaviors related to two core symptom domains, despair and anhedonia, including a longer immobility time in the forced swim test (FST) and a lower sucrose intake in the sucrose preference test (SPT) when compared with F0-Ctl (Fig. 1, C and D). Development of this trait was not due to an alteration in locomotor activity because F0-Dep covered a total distance similar to that of F0-Ctl in an open-field test (fig. S2A). Moreover, the plasma levels of corticosterone, a key hormone in the hypothalamic-pituitary-adrenal (HPA) axis and a well-characterized marker of stress intensity (21, 25), were markedly elevated in F0-Dep (Fig. 1E).

Depression results from maladaptive stress-induced changes in molecular, cellular, and synaptic neurotransmission in various brain regions and discrete neural circuits (7). The paraventricular nucleus of the hypothalamus (PVN), the crucial component of the HPA axis, showed elevated mRNA expression of corticotropin-releasing hormone (CRH) in F0-Dep (Fig. 1F), which was consistent with the increase in the plasma corticosterone level. These results together indicate an excessive HPA axis activation. In addition, the hippocampus (7, 26), medial prefrontal cortex (mPFC) (26, 27), and lateral habenula (LHb) (28–30) are involved in the pathophysiology of depression and jointly constitute an evolutionarily conserved core neural circuit that is essential for flexible and adaptive behavioral responses to environmental conditions and internal states (31). In particular, dysfunction of glutamatergic neurotransmission (32) and loss of neurotrophic factors (6) have been frequently documented in the hippocampus and mPFC of depressed patients. Analyses of the gene expression signatures in the hippocampus and mPFC of F0-Dep revealed aberrant expression of glutamate signaling genes (down-regulation of GluA1 and GluA2 and up-regulation of GluN2A and/or GluN2B) and reduced expression of synaptic plasticity genes (αCamKII, βCamKII, and ARC), synaptic structural genes (calmodulin-2, synapsin-3, and Rab3A), and neurotrophic factor genes [brain-derived neurotrophic factor (BDNF), glial cell line–derived neurotrophic factor (GDNF), nerve growth factor (NGF), vascular endothelial growth factor A (VEGFA), and VEGFB] (Fig. 1F). In contrast, βCamKII expression was notably up-regulated in LHb (Fig. 1F), which is also consistent with a previous report (30). Immunoblotting analysis validated the aberrant expression of glutamate receptor subunits and signaling proteins mediating synaptic transmission and plasticity in the hippocampus and mPFC of F0-Dep (Fig. 1G).

Paternal transmission of stress-induced depression-like symptoms to offspring

Next, we designed experimental strategies (33) to investigate whether depressive traits in fathers are inherited by the offspring. Briefly, F0-Dep and F0-Ctl were separately mated with healthy females, and their F1 offspring were screened for depression-like symptoms after becoming adults (Fig. 1H; detailed experimental design is shown in fig. S1). Despite being born to F0-Dep, the F1 offspring of depression-like model (F1-Dep) grew normally and had the same weight gain as F1 control offspring (F1-Ctl) (Fig. 1I). Under baseline conditions, F1-Dep also showed an immobility time and sucrose intake similar to those of F1-Ctl (Fig. 1, J and K). However, intriguingly, after exposure to a slight chronic variable stress (CVS) lasting for 2 weeks (34), F1-Dep recapitulated the paternal depressive-like phenotypes, including a significantly longer floating time and less sucrose consumption, whereas F1-Ctl did not express depressive-like behaviors (Fig. 1, J and K). The open-field test confirmed that these behavioral phenotypes in F1-Dep were independent of locomotor activity (fig. S2B). In addition, plasma corticosterone levels were remarkably increased in F1-Dep (Fig. 1L). To validate the relevance of environmental stimuli for the incidence of depression-like symptoms in offspring, we gradually strengthened the CVS procedure from 1 to 3 weeks. While 1 week of CVS was not sufficient to induce increased immobility and decreased sucrose consumption in F1-Dep, 2 or 3 weeks of CVS generated an apparent depression-like behavior in F1-Dep (fig. S2, C and D). Moreover, the behavioral assays were separately assessed on F1 males and females, and both genders exhibited a similar tendency to acquire the depression-like phenotypes after CVS exposure (fig. S2, E and F). The transmission of stress vulnerability across generations was also observed in another depression-like model established by chronic restraint stress (CRS) (fig. S3, A and B). Similar to the CMS model, the F1 offspring (F1-Dep) born to F0 males of depression-like model (F0-Dep) exhibited normal behavioral performances under baseline conditions (fig. S3, C to E) but were more vulnerable to depressive-like behaviors than F1-Ctl when exposed to a slight CVS (fig. S3, C to E). These results indicate that the F1 offspring of F0-Dep display reduced resilience to stress and enhanced susceptibility to depression-like phenotypes, although they appear normal under baseline conditions.

To explain the observed depression-like behaviors in F1-Dep, we performed molecular profiling in several key brain regions related to depression. F1-Dep had gene expression signatures similar to those of F0-Dep when exposed to CVS, characterized by overexpression of CRH mRNA in the PVN; dysregulation of mRNAs of glutamate receptors, synaptic proteins, and neurotrophic factors in the hippocampus and mPFC; and up-regulation of GluA1/2, Rab3A, BDNF, and βCamKII mRNAs in the LHb (Fig. 1M and fig. S4A). Immunoblotting analysis confirmed the aberrant expression of glutamate signaling and synaptic proteins in the hippocampus and mPFC of F1-Dep (Fig. 1N). Furthermore, genome-wide RNA sequencing was performed to capture transcriptome alterations in the hippocampus of F0-Dep and F1-Dep compared with those in nonstressed F0-Ctl and F1-Ctl. Rank-rank hypergeometric overlap (RRHO) analysis, used to identify patterns and strengths of genome-wide overlap in a threshold-free manner, indicated a substantial overlap of the up-regulated and down-regulated hippocampal genes between F0-Dep versus F0-Ctl and F1-Dep versus F1-Ctl (Fig. 1O). Hierarchical clustering also revealed similar hippocampal gene profiles in F0-Dep and F1-Dep, while the gene profile in F0-Ctl and F1-Ctl was in a distinct cluster group (Fig. 1P). Then, a gene ontology (GO) clustering analysis was performed to look for biological processes that might be associated with these altered genes. Among the 10 top-ranked GO clusters in each group, 6 GO clusters are shared between F0-Dep versus F0-Ctl and F1-Dep versus F1-Ctl, and these overlapped GO functional categories are directly related to “nervous system development,” “synaptic signaling,” “localization, transport,” “regulation of signaling,” “cognition, behavior,” and “locomotion” (Fig. 1Q and table S1). These data strongly suggest that F1 offspring born to F0-Dep may have impaired neural function as a result of maladaptive molecular and signaling changes and are therefore more susceptible to stress-induced depression-like symptoms.

Abnormal neuronal activation and synaptic transmission in F0 males of depression-like model is conferred to offspring

To dissect the functional changes in the hippocampus, mPFC, and LHb, we assessed neuronal activation and synaptic transmission by c-Fos immunocytochemistry, a sensitive marking technique for neuronal activation, and whole-cell patch-clamp recordings on brain slices. In accordance with the increase in CRH mRNA and plasma corticosterone, CRHergic neurons were significantly activated in the PVN of F0-Dep compared with those of F0-Ctl (Fig. 2, A and D, and fig. S5A). Accordingly, CRHergic neurons were apparently activated in the PVN of F1-Dep compared with those of F1-Ctl (Fig. 2, A and D, and fig. S5A), suggesting a significant increase in HPA axis activity in F1-Dep. Likewise, an apparent increase in neuronal activation was also observed in the LHb of F1-Dep (fig. S4, B and C). However, neuronal activation was markedly silenced in the hippocampus and mPFC of F0-Dep compared with that in F0-Ctl (Fig. 2, B, C, E, and F, and fig. S5, B and C). Accordingly, a pronounced decrease in neuronal activation was observed in the hippocampus and mPFC of F1-Dep compared with that in F1-Ctl (Fig. 2, B, C, E, and F, and fig. S5, B and C). These results are consistent with the notion that depression is associated with reduced neuronal activation in the hippocampus and mPFC, whereas hyperactivity of LHb and PVN plays a causal role in depression (7, 26, 27, 29, 30). Furthermore, both the frequencies and amplitudes of spontaneous excitatory postsynaptic currents (sEPSCs) were significantly decreased in the hippocampal and mPFC pyramidal neurons of F0-Dep compared with those in F0-Ctl (Fig. 2, G and H, top), and a similar degree of decline was observed in the hippocampal and mPFC pyramidal neurons of F1-Dep compared with those in F1-Ctl (Fig. 2, G and H, bottom). These results suggest that the synaptic transmission and neuronal activity profiles in the neural circuits of F1-Dep were similar to those of F0-Dep despite the fact that F1-Dep were reared normally and did not undergo stress exposure like F0-Dep.

Behavioral performances of F2 offspring born to F1 offspring of depression-like model

Subsequently, we determined whether the depressive-like traits would be transmitted to the F2 generation. Males of F1-Dep and F1-Ctl were bred with normal females, and the F2 offspring (F2-Dep versus F2-Ctl) were examined (fig. S6A; detailed experimental design is shown in fig. S1). Compared with F2-Ctl, F2-Dep showed no susceptibility to a CVS stimulus (fig. S6, B and C), indicating an intergenerational rather than transgenerational inheritance of depression susceptibility. These results are consistent with previous observations that mammals rarely have transgenerational epigenetic inheritance, whereas in plants, worms, and yeast, phenotypic transmission can span multiple generations (9, 14).

Sperm sRNAs contribute to paternal transmission of depression-like symptoms

Such father-to-offspring transmission excludes the maternal effects and suggests the vital position of the paternal germ line as the carrier of intergenerational information. Increasing evidence suggests that paternal characteristics acquired during environmental exposures can be inherited by future generations through the sperm (9, 10, 13), yet the active components of sperm that interlink paternal environment and offspring outcomes remain to be elucidated. Sperm RNA, previously considered to be negligible remnants of spermatogenesis, was recently shown to be transmitted to the zygotes during fertilization (16, 18, 21, 35). To assess whether sperm RNA is associated with the increased incidence of depression-like phenotypes in offspring, we purified total RNAs from the sperm of F0-Dep or F0-Ctl mice and injected them into normal zygotes (RNA injection was normalized to the amount of approximately 10 sperm) (16, 18), and then, the embryos were transferred into surrogate mothers to generate offspring (RNA-Dep versus RNA-Ctl); diethylpyrocarbonate (DEPC) water instead of sperm was processed for RNA isolation and injected into normal zygotes, and the resulting offspring served as a mock control (fig. S7A; detailed experimental design is shown in fig. S1). RNA-Ctl exhibited no difference in behaviors compared with mock control (fig. S7, B and C), indicating that the injection of sperm RNA from F0-Ctl into zygotes did not affect offspring phenotypes. While RNA-Dep displayed no obvious depressive-like behaviors under baseline conditions in comparison with RNA-Ctl (fig. S7, B and C), RNA-Dep developed significant depressive-like behaviors in response to CVS (fig. S7, B and C). Overall, injection of sperm RNA into zygotes produced the same depressive-like behavioral alterations in the resulting offspring as those born to stress-induced F0-Dep.

To narrow the active components in sperm RNA, we specifically enriched small RNA (sRNA; <200 nt) fraction from sperm of F0-Dep and F0-Ctl and microinjected into wild-type zygotes to generate in vitro fertilization (IVF) offspring (sRNA-Dep versus sRNA-Ctl; sRNA injection was normalized to the amount of approximately 10 sperm), and equal amount of synthetic scrambled RNA (scrRNA; random sequences and 0 to 50 nt in length) was processed for RNA isolation instead of sperm and injected into zygotes to generate mock control offspring (Fig. 3A; detailed experimental design is shown in fig. S1). While sRNA-Dep, sRNA-Ctl, and mock control exhibited same behavioral performances under baseline conditions (Fig. 3, B and C), and sRNA-Ctl and mock control showed similar behavioral performances after exposure to CVS (Fig. 3, B and C), sRNA-Dep were more susceptible to depression-like behaviors induced by CVS, including a longer immobility time in FST and a lower sucrose intake in SPT (Fig. 3, B and C). Compared with sRNA-Ctl, sRNA-Dep showed abnormal activation of the HPA axis, including elevated plasma corticosterone levels (Fig. 3D), stimulated CRH expression (Fig. 3E), and increased activation of CRHergic neurons in the PVN (Fig. 3F and fig. S8A). Likewise, a pronounced increase in depression-related genes and neuronal activation was observed in the LHb of sRNA-Dep compared with sRNA-Ctl (fig. S9). Furthermore, aberrant expression of depression-related genes/proteins (Fig. 3, G, H, and J) and decreased neuronal activation (Fig. 3, I and K, and fig. S8, B and C) were observed in the hippocampus and mPFC of sRNA-Dep compared with sRNA-Ctl. Moreover, both the frequency and amplitude of sEPSCs were decreased in the hippocampal and mPFC pyramidal neurons of sRNA-Dep (Fig. 3, L and M). In addition, we specifically enriched lRNA (> 200 nt) fraction from sperm of F0-Dep and F0-Ctl and microinjected it into wild-type zygotes to generate IVF offspring (lRNA-Dep versus lRNA-Ctl) (fig. S10A). lRNA-Dep did not display apparent depression-like behaviors compared with lRNA-Ctl both under baseline conditions and after CVS exposure (fig. S10, B and C). These results demonstrate that sperm sRNAs rather than lRNAs contribute to the effect of sperm RNAs on inducing depressive-like symptoms in offspring.

sRNA profiles are differentially expressed in sperm after exposure to stress

Sperm carry a population of sRNAs, including miRNAs, PIWI-interacting RNAs (piRNAs) and tsRNAs (36, 37). To identify which specific subtypes of sperm sRNAs cause abnormalities in offspring, we examined the sRNA profiles of sperm derived from F0-Dep and F0-Ctl by RNA deep sequencing. Length distribution analysis revealed similar sRNA compositions in sperm from F0-Dep and F0-Ctl (fig. S11A). Analysis of differentially expressed sRNAs using a stringent threshold (mean reads > 500, fold change > 2, and P < 0.05) identified 19 miRNAs, 24 piRNAs, and 0 tsRNAs that were significantly altered in F0-Dep sperm compared with those in F0-Ctl sperm (Fig. 4A and tables S2 and S3). A larger proportion of miRNAs (18 up-regulated versus 1 down-regulated) showed increased expression in F0-Dep sperm, while most piRNAs (6 up-regulated versus 18 down-regulated) exhibited a decreasing trend (Fig. 4A). Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed the accuracy of RNA deep sequencing. In total, 16 and 1 miRNAs were validated to be significantly up- and down-regulated in F0-Dep sperm, respectively; 1 and 5 piRNAs were significantly up- and down-regulated in F0-Dep sperm, respectively; and 0 tsRNAs were significantly altered in F0-Dep sperm (Fig. 4B). Intriguingly, when the 19 miRNAs were assessed in the sperm of F1-Ctl and F1-Dep (under baseline condition without CVS stimulus), no miRNA was found to be significantly changed in F1-Dep sperm (fig. S11C). These results are consistent with the observation that the depressive-like traits could not be transmitted from F1 to the F2 generation. In summary, the above results illustrate that sRNAs are indeed very sensitive to paternal stress experience and are differentially expressed in sperm after exposure to stress.

Behavioral, neuronal, and gene alterations of IVF offspring born to zygotes injected with synthetic miRNAs

Because miRNAs have a well-established regulatory role in embryonic development, inherited miRNAs are hypothesized to reshape offspring development toward depression-like phenotypes. Thus, a subset of synthetic miRNAs mimicking the 16 most highly expressed sperm miRNAs in F0-Dep or equal amounts of scrRNA were injected into normal zygotes (miRNAs were injected at levels equivalent to natural conditions), and the IVF offspring were assessed for depression-like phenotypes (miRNA-Dep versus scrRNA-Ctl) (fig. S12A; detailed experimental design is shown in fig. S1). Although no behavioral alterations (fig. S12, B and C) were exhibited by the offspring under baseline conditions, miRNA-Dep had a higher vulnerability to depression-like behaviors in response to CVS (fig. S12, B and C). The plasma corticosterone levels were also higher in miRNA-Dep than in scrRNA-Ctl (fig. S12D). Moreover, neuronal activation in depression-related brain regions of miRNA-Dep was also reshaped to an abnormal state (fig. S12, E to G). In addition, miRNA-Dep had a neuroelectrophysiological phenotype similar to that of F0-Dep (fig. S12, H and I). A portion of marker genes recapitulating depression signatures was also aberrantly expressed in miRNA-Dep, including overexpression of CRH in PVN and down-regulation of some glutamate receptors, synaptic plasticity genes, and neurotrophic factors in the hippocampus and mPFC (fig. S12J). Thus, miRNA-mediated epigenetic mechanisms might at least partially contribute to the intergenerational transmission of stress-induced depression vulnerability.

Adjusting the imbalance of miRNAs in zygotes rescues the abnormalities in the F1 offspring born to F0 males of depression-like model

To investigate whether depression inheritance is mediated causally via a specific group of miRNAs, we first fertilized normal oocytes by sperm from F0-Dep or F0-Ctl, and then, a combination of miRNA antisense strands designed to block the 16 increased sperm miRNAs was injected at the zygote stage to neutralize the effects of inherited sperm miRNAs and generate IVF offspring (F1-Dep + Anti or F1-Ctl + Anti); for the control groups, normal oocytes were fertilized by sperm from F0-Dep or F0-Ctl and then injected with scrRNA to generate IVF offspring (F1-Dep + scrRNA or F1-Ctl + scrRNA) (Fig. 4C; detailed experimental design is shown in fig. S1). Under baseline conditions, there were no differences in behavioral performances between these four groups (fig. S13, A and B). After CVS exposure, F1-Dep + scrRNA exhibited considerable depression-like behaviors compared with F1-Ctl + scrRNA, whereas F1-Dep + Anti displayed relatively normal behavioral performances nearly equal to those in F1-Ctl + scrRNA (Fig. 4, D and E). As a control, F1-Ctl + Anti did not display depressive-like behaviors compared with F1-Ctl + scrRNA (Fig. 4, D and E), indicating that injection of miRNA antisense strands into zygotes alone had no obvious effect on offspring phenotypes. Likewise, F1-Ctl + Anti and F1-Ctl + scrRNA had similar plasma corticosterone levels, whereas the high plasma corticosterone levels in F1-Dep + scrRNA were significantly decreased in F1-Dep + Anti (Fig. 4F). Moreover, while F1-Ctl + Anti displayed normal neuronal activation and synaptic transmission compared with F1-Ctl + scrRNA, the aberrations of neuronal activation and synaptic transmission in the PVN, hippocampus, and mPFC of F1-Dep + scrRNA were significantly attenuated in F1-Dep + Anti and recovered to nearly normal states as compared with F1-Ctl + scrRNA (Fig. 4, G to J, and fig. S13, C to E). To validate again that the miRNA antisense strands can counteract the vulnerability to depression-like phenotypes triggered by inherited miRNAs, we compared offspring born to zygotes coinjected with miRNA antisense strands and sperm RNA from F0-Dep (RNA-Dep + Anti) with those with sperm RNA from F0-Dep or F0-Ctl along with scrRNA (RNA-Dep + scrRNA or RNA-Ctl + scrRNA) (fig. S14A). Offspring of these three groups had similar behavioral responses under baseline conditions (fig. S14, B and C). While RNA-Dep + scrRNA exhibited increased risk for depression-like behaviors compared with RNA-Ctl + scrRNA, RNA-Dep + Anti were not susceptible to depression-like behaviors under a CVS stimulus (fig. S14, B and C). Accordingly, the increased plasma corticosterone levels and abnormal neuronal activation and synaptic transmission in RNA-Dep + scrRNA were restored to normal states in RNA-Dep + Anti (fig. S14, D to L).

Sperm sRNAs from depression-like model reprogram early embryonic transcriptional profiles

We further investigated the mechanism by which sperm sRNAs contribute to depression inheritance. Because the early embryonic period represents a window of plasticity important for adult phenotypes, we injected sperm sRNAs from F0-Dep or F0-Ctl into zygotes and assessed miRNA alterations when the embryos developed to the E3.5 (embryonic day 3.5) blastocyst stage (sRNA-Dep-E3.5 versus sRNA-Ctl-E3.5). Among the 16 overexpressed miRNAs in the sperm of F0-Dep, 13 exhibited a 1.5- to 4-fold increase in sRNA-Dep-E3.5 embryos (fig. S15A). Furthermore, when we injected synthetic mimics of 16 miRNAs or equal amounts of scrRNAs into normal zygotes and assessed miRNA alterations in the E3.5 blastocyst stage (miRNA-Dep-E3.5 versus scrRNA-Ctl-E3.5), 15 were found to have a two- to sixfold increase in miRNA-Dep-E3.5 embryos (fig. S15B). To investigate the potential impact of increased miRNAs on embryonic development, we assessed alteration of gene profiles by single-cell transcriptome RNA sequencing in sRNA-Dep-E3.5 versus sRNA-Ctl-E3.5. A total of 264 (107 up-regulated and 157 down-regulated) embryonic genes were identified as differentially expressed (fold change > 2 and P < 0.05) in sRNA-Dep-E3.5 (Fig. 5A). GO analysis of these differentially expressed genes identified enrichment of GO clusters whose dysfunction frequently contribute to neuropsychiatric abnormalities, such as synaptic signaling, neuronal differentiation, and neuron development (Fig. 5B and table S4). We assessed whether these differentially expressed genes were potentially regulated by the 17-miRNA set (16 up-regulated miRNAs and down-regulated miR-184). In total, 78 genes (1 up-regulated and 77 down-regulated) among the 264-gene set had the tendency to be directly targeted, and this number is significantly higher than that obtained under random simulations (Fig. 5C). Through literature mining, many genes among the 78-gene set were identified to be implicated in the modulation of neuronal function and pathophysiology (e.g., synaptic plasticity, dendritic spine formation, and nerve outgrowth) (30, 38–40). Among these putative miRNA-target pairs, App, Tspan7, Wnk3, Ly6a, Grin3a, and βCamkII were selected as the representative and specially characterized. Embryonic stem (ES) cells transfected with synthetic miRNA mimics showed reduction of these six proteins (fig. S15C), and a luciferase reporter assay confirmed direct binding of corresponding miRNAs to the 3′ untranslated regions (3′UTRs) of the six genes (fig. S15D). Theoretically, these neuronal genes that originally tend to be fine-tuned and precisely controlled in early embryonic period might be inappropriately disrupted and reprogrammed by inherited sperm miRNAs. In line with this hypothesis, aberrant expression of these genes was observed in developing embryos of F1-Dep versus F1-Ctl and sRNA-Dep versus sRNA-Ctl. In detail, while the expression levels of App, Tspan7, Wnk3, Ly6a, and Grin3a increased sharply from the four-cell stage to the morula stage in F1-Ctl embryos, the elevation of these genes was significantly delayed in F1-Dep embryos (Fig. 5D). In contrast, while βCamkII expression was maintained at a basal level in F1-Ctl embryos, its expression was markedly induced in F1-Dep embryos (Fig. 5D). Consistently, the altered gene profiles in early embryos of sRNA-Dep versus sRNA-Ctl recapitulated those of F1-Dep versus F1-Ctl (Fig. 5E). The results suggest that sperm sRNAs may imprint perturbations in the core neuronal circuits during early embryonic development.

Study design

The objective of this study was to investigate whether paternal depression-like phenotypes acquired by stress exposure can be inherited by offspring via a causal role of miRNAs in the sperm. To confirm whether depressive-like traits in fathers are inherited by the offspring, male C57BL/6J littermates (8 weeks old) were randomly assigned into two groups: One group (F0-Dep) was used to establish a CMS-induced depression-like model by exposing male mice to unpredictable mild stresses daily for 5 weeks (detailed protocol of CMS is listed in table S5), and the other group (F0-Ctl) was set as nonstressed controls. Immediately after the CMS procedure, a portion of the stressed F0-Dep and nonstressed F0-Ctl was screened for body weight, behavioral performances (open field → SPT → FST), corticosterone levels, neuronal activation, and depression-related gene alteration to validate the successful establishment of depression-like model, and the rest of F0-Dep and F0-Ctl were separately mated with normal females to generate F1 offspring (F1-Dep and F1-Ctl). F1-Ctl and F1-Dep were also randomly assigned into different testing groups and separately screened for behavioral performances (open field → SPT → FST), corticosterone levels, neuronal activation, and hippocampal gene profiles under baseline conditions or were screened for behavioral performances and depression-related gene alteration after 2 weeks of CVS (detailed protocol of CVS is listed in table S5). To obtain F2 offspring, male F1-Ctl and F1-Dep were separately mated with normal females. Furthermore, four IVF approaches were conducted to causally test whether depressive-like traits in fathers are inherited by the offspring through the sperm, sperm total RNA, sperm sRNA, or sperm miRNA. Behavioral performances (SPT → FST), corticosterone levels, neuronal activation, and depression-related gene alteration were measured in the same way as mentioned above. All procedures were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Ethical Committee of Nanjing University. Investigators were blinded to group assignment during behavioral experiments.

Mice

Mice of the inbred C57Bl/6J strain were obtained from the National Resource Center of Model Mice (NRCMM) and Model Animal Research Center of Nanjing University (MARC) (Nanjing, China) and maintained on a 12-hour light/dark cycle (lights on at 7:00 a.m.) with free access to food and water, unless indicated.

Stress-induced depression-like models

Behavioral tests

Corticosterone assessment

To measure basal corticosterone levels, submandibular blood samples were obtained directly from the home cage condition, using animal lancets, from 7:00 to 9:00 a.m. Blood samples were collected into EDTA-coated tubes, and plasma was isolated after centrifugation of the blood samples at 2000g for 20 min at 4°C. Corticosterone in 10 μl of plasma was measured using an enzyme-linked immunosorbent assay kit (Enzo Life Sciences, NY) according to the manufacturer’s instructions. Three replicates were measured for each sample.

Breeding scheme

RNA isolation and quantitative RT-PCR analyses of mRNA, miRNA, tsRNA, and piRNA

Total RNA was isolated from tissues and sperm using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. For quantitative RT-PCR analysis of mRNA, 1 μg of total RNA was reverse-transcribed to complementary DNA (cDNA) using AMV reverse transcriptase (TaKaRa, Dalian, China) and oligo(dT) primer (TaKaRa). Quantitative RT-PCR was performed using a SYBR Green PCR kit on an Applied Biosystems 7300 Sequence Detection System. Analysis of gene expression was performed using the △△C_T method, and relative gene expression was normalized to β-actin mRNA levels. Three replicates were measured for each sample. Primer sequences are listed in table S6.

For miRNA analysis (59), 100 ng of total RNA was reverse-transcribed to cDNA using AMV reverse transcriptase (TaKaRa) and stem-loop RT primer (Applied Biosystems). Quantitative RT-PCR was performed using a TaqMan PCR kit on an Applied Biosystems 7300 Sequence Detection System. The relative miRNA expression was normalized to U6 small nuclear RNA (snRNA). Three replicates were measured for each sample.

For tsRNA and piRNA analyses (18), 100 ng of total RNA was reverse-transcribed to cDNA using the miScript II RT Kit (QIAGEN, Hilden, Germany). In a reverse transcription reaction with miScript HiFlex Buffer, piRNA or tsRNA is first polyadenylated by poly(A) polymerase and converted into cDNA by reverse transcriptase with universal RT primer. Subsequently, quantitative PCR was performed using the miScript SYBR Green PCR Kit (QIAGEN) with a specific forward primer and a universal reverse primer (primer sequence is listed in table S6). After the reaction, the lengths of the RT-PCR products were elongated. The relative expression of tsRNA and piRNA was normalized to U6 snRNA. Three replicates were measured for each sample. To validate the specificity of each primer set, no-template controls for each piRNA and tsRNA were assessed simultaneously. In addition, the PCR products was confirmed by electrophoresis and by TA cloning and sequencing. A total of 24 piRNAs and 5 tsRNAs were correctly and efficiently amplified by quantitative RT-PCR, and the no-template controls amplified at a much higher cycle range than these piRNAs and tsRNAs (fig. S16).

Western blot analysis

Samples from tissues and cells were sonicated in radioimmunoprecipitation assay buffer [0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, and 50 mM tris-HCl (pH 7.5) with phenylmethylsulfonyl fluoride] (1:100, Beyotime Biotechnology, China). The samples were centrifuged at 12,000g for 10 min at 4°C, and the supernatant was retained for Western blot analysis. The bicinchoninic acid (BCA) colorimetric method was used to determine the total amount of protein. Equal amounts of protein (30 to 60 μg) were resolved via 10 to 12.5% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Detection was based on a fluorescent secondary antibody that was visualized using the Tanon 5200 Multi detection system (Tanon, Shanghai, China). The data were quantified using ImageJ software (NIH, Bethesda, MD), and the relative protein expression was normalized to the value of β-actin. Information on primary antibodies is listed in table S7.

To conduct analyses on five different proteins (GluA1, GluA2, GluN2A, GluN2B, and CamkII) and one internal control (β-actin) in a limited amount of hippocampus and mPFC tissues with six different antibodies, sliced bands from the same blot were used in this study. On the basis of the apparent molecular weight of GluA1, GluA2, GluN2A, GluN2B, CamkII, and β-actin (~110, ~115, ~175, ~180, ~65, and 42 kDa) (fig. S17), the PVDF membrane was cut (at 70- and 130-kDa marker) into three parts (0 to 70 kDa, 70 to 130 kDa, and 130+ kDa). The three parts were then first blotted with GluN2A (175 kDa), GluA1 (110 kDa), and β-actin (42 kDa) antibodies, respectively, and detected with a fluorescent secondary antibody. Both primary and secondary antibodies were then removed using antibody removal solution (Beyotime Biotechnology), blocked again, and blotted with GluN2B (180 kDa), GluA2 (115 kDa), and CamkII (65 kDa) antibodies, respectively. The same experiment was repeated three times, and in each biological repeat, all sliced membranes are stripped only once (blotted twice). It did happen, however, that some antibodies did not work well because of repeated membrane washing, in which case we would rerun the SDS-PAGE and reblot with the antibody of trouble together with its own β-actin control (and that is why more than one internal control was shown for one set of experiment).

Immunofluorescence

Immunofluorescence was performed as previously reported (23, 60). Briefly, mice were deeply anesthetized with sodium pentobarbital (65 mg/kg) and perfused transcardially with 20 ml of normal saline, followed by 100 to 150 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. The brain was gently removed, trimmed, and postfixed in the same fixative for 12 hours at 4°C and then cryoprotected with 30% sucrose for 48 hours. Frozen coronal sections (20 μm in thickness) containing the PVN, hippocampus, or mPFC were acquired by a freezing microtome (CM3050S, Leica, Wetzlar, Germany) and mounted on gelatin-coated slides. The slices were rinsed with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 and then incubated in 10% normal bovine serum in PBS containing 0.1% Triton X-100 for 30 min. Sections were incubated overnight at 4°C with the following primary antibodies: rabbit anti–c-Fos, chicken anti-NeuN, and goat anti–corticotropin-releasing factor. After a complete wash in PBS, the sections were incubated in the appropriate secondary antibodies (1:2000, Invitrogen) for 2 hours at room temperature in the dark. The slices were washed and mounted in Fluoromount-G mounting medium (SouthernBiotech, Birmingham, AL). Incubations replacing the primary antiserum with control immunoglobulins and/or omitting the primary antiserum were used as negative controls. The micrographs were taken with a TCS SP8 inverted laser scanning confocal microscope (Leica). Digital images from the microscope were recorded with LAS X Viewer software (Leica). Cell counts were performed using Image-Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD) in combination with manual scoring to ensure accuracy. All counting was performed by an investigator who was blinded to the experimental group. Information on primary antibodies is listed in table S7.

Brain slice preparation and whole-cell patch-clamp recordings

Adult mice (25 to 30 g, 10 to 12 weeks) were used for whole-cell recordings. After the mouse was decapitated under sodium pentobarbital (40 mg/kg) anesthesia, the brain was quickly removed and cooled in ice-cold standard artificial cerebrospinal fluid (ACSF; composition: 124 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 1.3 mM MgSO₄, 26 mM NaHCO₃, 2 mM CaCl₂, and 10 mM d-glucose) saturated with 95% O₂/5% CO₂. Coronal slices (300 μM in thickness) containing the hippocampus or mPFC were cut with a vibroslicer (VT 1200S, Leica Microsystem, Wetzlar, Germany) according to the mouse brain atlas. The slices were stored for at least 1 hour in a holding chamber filled with 95% O₂ and 5% CO₂ oxygenated ACSF at 35 ± 0.2°C before any experiment was started.

Whole-cell patch-clamp recordings were performed as previously reported (23, 60). All EPSC recordings were made with electrodes filled with a solution containing the following: 120 mM cesium methanesulfonate, 20 mM CsCl, 10 mM Hepes, 0.2 mM EGTA, 10 mM sodium phosphocreatine, 5 mM QX-314, 4 mM Na₂–adenosine 5′-triphosphate, and 0.4 mM Na–guanosine 5′-triphosphate (pH 7.2) (290 to 330 mOsm). The electrode resistance in the bath solution was 5 to 7 megohms, and series resistance (< 25 megohms) was monitored continuously to ensure stability within 20%. Whole-cell patch-clamp recordings were performed on the pyramidal neurons, the principle neurons in the hippocampal area CA3 and the prelimbic (PrL) area of mPFC using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), and data were filtered at 2 kHz and digitized at 5 kHz using pClamp10 software. The sEPSCs were recorded at a holding potential (−65 mV) in the presence of the SR95531 (20 μM; Tocris Bioscience, Bristol, UK), which was used to block γ-aminobutyric acid type A receptor–mediated inhibitory postsynaptic currents.

RNA sequencing

Sperm sample collection and sRNA sequencing

Microinjection of sperm RNA, sperm sRNA, sperm lRNA, and synthetic RNA and transfer of embryos

Total RNA was extracted from sperm using TRIzol Reagent (Invitrogen). sRNA and lRNA were extracted from sperm using the MirVana Protein and RNA Isolation System (Ambion, Austin, TX). Synthetic miRNA mimics, antisense strands, and scrRNA were purchased from GenePharma (Shanghai, China). According to the information provided by the manufacturer, miRNA mimics are synthetic double-stranded RNA oligonucleotides that specifically mimic endogenous mature miRNAs; they are designed and modified to ensure that the correct strand, representing the desired mature miRNA, is taken up into the RNA-induced silencing-like complex responsible for miRNA activity. MiRNA antisense strands are single-stranded, chemically modified oligonucleotides designed to specifically bind to and inhibit endogenous mature miRNAs. MiRNA antisense strands share exact complementarity with the miRNA and can form a tight duplex with the miRNA guide strand, therefore preventing the miRNA from binding to its intended target because of the steric blocking. Sequences of synthetic miRNA mimics and antisense strands are listed in table S8.

Total RNA, sRNA, or lRNA was adjusted to a concentration of 2 ng/μl. Equal amounts of 16 chemically synthetic miRNA mimics and/or antisense strands were mixed and adjusted to a concentration of 2 ng/μl (each miRNA mimic or antisense strand is 0.125 ng/μl). This amount (2 ng/μl) is approximately equal to the total RNA of 10 sperm according to previous reports (16, 18). All RNAs were microinjected into zygotes of the C57Bl/6J background using a Leica microinjection system. The zygotes were then cultured in M16 (Sigma-Aldrich) at 37°C in 5% CO₂. The two-cell embryos were transferred to the oviduct of the surrogate mother of the ICR background. The offspring of each group (RNA-Dep versus RNA-Ctl, sRNA-Dep versus sRNA-Ctl, lRNA-Dep versus lRNA-Ctl, and miRNA-Dep versus scrRNA-Ctl) were screened for behavioral performances, corticosterone levels, neuronal activation, and depression-related gene alteration until adulthood.

Approximately 300 embryos were injected for each condition, and 80 to 85% implanted embryos survived up to E3.5, 30 to 35% to E5.5, and 30 to 35% until adulthood. There was no significant difference in the embryo survival rate between the depression-like model and control groups in each developmental stage.

Early embryo collection and single-cell transcriptome RNA sequencing

miRNA target prediction, cell transfection, and luciferase reporter assay

Data statistics

All statistical tests were performed under the open-source statistics package R or using GraphPad Prism software 7 (San Diego, CA). Normality and equal variances between group samples were assessed using Shapiro-Wilk test and Brown-Forsythe tests, respectively. When normality and equal variance were achieved between sample groups, one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test (Figs. 1J; 3B; and 4, D and F to J), two-way ANOVA followed by Bonferroni’s multiple comparisons test (Fig. 1, B and I), t test (Figs. 1, C, E, and L; 2, G and H; and 3, D to F, I, and K to M), or multiple t test (Figs. 1, F, G, M, and N; 2, D to F; 3, G, H, and J; 4B; and 5, D and E) was used. Where normality or equal variance of samples failed, Kruskal-Wallis one-way ANOVA followed by Dunn’s correction (Figs. 1K, 3C, and 4E) or Mann-Whitney U test (Fig. 1D) was performed. Differences are considered statistically significant at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.