LPS maternal stimulation reduces the reproductive performance of laying hens and their offsprings
To explore the possible intergenerational transmission effects caused by the LPS maternal stimulation, we first measured the egg-laying rate of F0 hens. The results showed that the total egg-laying rate of the three weeks post the LPS treatment was 17.17% lower than that of the two weeks before the treatment. Also, the total egg-laying rate of two weeks before the LPS treatment was 3.20% higher than that of the control group with non-significant differences (Fig. 2A, P > 0.05). However, the egg-laying rate of LPS group became 8.31% significantly lower than that of the controls in the three weeks after the treatment (Fig. 2A, P < 0.05). The difference between the two groups widened to 11.51% after the stimulation, indicating that LPS stimulation could significantly reduce the reproductive performance of laying hens. Furthermore, the daily egg-laying rate of the LPS group was significantly lower than that of the control group (Fig. 2B, P < 0.05), which proved that LPS treatment could significantly reduce the reproductive performance of the laying hens. We further examined the two groups of hens as a whole union and calculated the differences in egg-laying rate between the two groups for 22 consecutive days and found that the daily egg-laying rate of the LPS group that was lower than the control group accounted for 72.73% of the total statistics (Fig. 2C), which was extremely significantly higher than the probability of the LPS group that was higher than that of the controls (18.18%, P < 0.001). These findings support the notion that LPS stimulation could reduce the egg-laying rate of hens.
For the aim of studying the effect of LPS maternal stimulation on the egg-laying rate of the corresponding untreated offspring, we calculated the daily egg-laying rate of F1 hens for 304 consecutive days (21-66W, except 39-40W), and found that the daily egg-laying rate of the F1 hens was significantly lower than that of the control group (Fig. 2D, P < 0.001). Furthermore, we compared the daily egg-laying rate between the two groups during the 304 days, and found that the egg-laying rate of the LPS group was lower than that of the control group almost during the entire laying period in the F1 generation (Fig. 2E), especially in the early and late laying stages. We also found that 90.79% (276/304) of the daily egg-laying rate in the LPS group was lower than that of the control, of which 30.07% were significantly different (Fig. 2E and 2F, P < 0.05). The daily egg-laying rate of the LPS group was significantly lower than that of the control group accounted for 28%, 18.25% and 54.76% in early, middle and late laying stages, respectively (Fig. 2F). These results showed that the LPS maternal stimulation has a greater negative impact on the early and late laying stages of F1 hens.
Intergenerational transmission of the transcriptome caused by LPS maternal stimuli
To reveal the molecular mechanism of LPS maternal stimulation that regulates the decreased egg-laying rate of the hens and their offspring from the perspective of immunity, we examined the transcriptome changes in the spleen using 53-week-old laying hens in F0 and F1 generations. It was found that LPS maternal stimulation caused changes in the F0 and F1 transcriptomes (Fig. 3A, B). Using the screening criteria of P <0.05 and FC > 1.5, 634 and 577 differentially expressed genes (DEGs) were obtained in F0 and F1, respectively, of which up-regulated genes in the LPS group accounted for 48.58% and 66.90%, respectively. With the criteria of P <0.05, FC > 2, 282 and 272 DEGs were separately detected in F0 and F1, of which up-regulated genes accounted for 50.35% and 59.56% (Fig. 3C). It can be seen that LPS activates the expression of some genes. The follow-up studies are based on P < 0.05, FC > 1.5.
To investigate the transcriptome's intergenerational transmission of maternal effect on laying hens, we examined the changes in the expression of common DEGs in the two generations of laying hens. The results showed that a total of 1077 DEGs were involved in the F0 and F1 generations, including 134 common DEGs (coDEGs), of which 63 coDEGs (47.01%, 42 up-regulated, 21 down-regulated) showed the same expression trend between the two generations (Fig. 3D). Furthermore, we used the FPKM values of the above 1077 DEGs to perform the correlation analysis between the two generations in each group. The results showed that the gene expression between the two generations of the same group had a very high positive correlation, and the correlation coefficients reached 0.9 (Fig. 3E, P < 2.2E-16), indicating that the effect of maternal stimulation on the transcriptome of laying hens has a transmission effect. We lists the 134 coDEGs between the two generations, including 63 coDEGs with the same expression trend, which can be used as marker genes for further studies, such as APLNR, BFSP1, C3, CCL1, CATHL2, CEP162, EXFABP, F13A1, and FOS (Fig. 3F).
The RNA-seq data used in this study was verified by RT-qPCR by randomly selecting three genes in the F0 and F1 generations. We compared the log2FC predicted by RNA-seq with the actual log2FC obtained by RT-qPCR. These results showed that all genes have the same expression trend (Fig. 4A). In addition, the regression analysis results showed that log2FC obtained by RNA-Seq is highly correlated with log2FC obtained by RT-qPCR (r = 0.92, P < 0.01), confirming the reliability of the current RNA-seq data (Fig. 4B).
LPS could influence the immune response of hens in F0 and F1 generations
To analyze the biological functions separately involved in the DEGs of F0 and F1 generations, we used Ingenuity Pathway Analysis (IPA) to predict Ingenuity Canonical Pathways using the DEGs of F0 and F1 generations. A total of 103 and 44 significantly enriched pathways were identified in F0 and F1 generations, respectively (Table S3 and S4, P < 0.05). Then 33 and 18 Ingenuity Canonical Pathways were separately retained with the screening criteria of P <0.01, of which 8 pathways were shared by both F0 and F1 generations. All of the eight pathways are directly or indirectly related to immune response. The eight shared pathways of F0 and F1 contained 34 and 23 genes, respectively, of which 15 genes were shared between the two generations, such as ALB, AMBP, C3, EGR1, F13A1, F2, FGA, FGB, FOS, GC, HPX, MST1, RBP4, SERPINC1, VTN (Table 1). These results indicated that the treatment of LPS could influence the immune system of the F0 generation, and the effect could be transmitted to the next generation.
We also used IPA to study upstream regulators of the DEGs in F0 and F1 generations and found that there were 6 (30%) shared upstream regulators in the top 20 upstream regulators of F0 and F1 generations, of which, all were related to immune-inflammatory responses, such as lipopolysaccharide, TNF, IL1B, PPARA, dexamethasone and IL6 (Table 2), further indicated that the immune system of F0 and F1 was affected. The discovery of a common regulator such as lipopolysaccharide suggests that the maternal response to LPS affects the immune system and has a long-lasting intergenerational transmission effect.
In order to further understand the intergenerational transmission effect of the LPS maternal stimulus, the 63 coDEGs with the same expression trend in F0 and F1 generations were used to perform GO term enrichment analysis. We only focused on GO terms related to biological processes, and top30 significantly enriched terms are shown in Fig. 5A. The majority of the top terms are related to immune system (from the bottom up), such as defense response to bacterium, inflammatory response, response to external stimulus, innate immune response, response to external biotic stimulus, immune response, humoral immune response, defense response to Gram-negative bacterium (Fig. 5A), suggesting that the LPS maternal stimulation could cause the immune-inflammatory response of the F0 generation, and the effect had been transmitted to the F1 generation. Further analysis of the seven terms (terms in red font) with FDR < 0.05 showed that these seven terms were related to immune-inflammatory response, including defense response to bacterium, response to external stimulus, antimicrobial humoral response, innate immune response and inflammatory response. In addition, we found that these seven terms were mainly associated with 10 DEGs such as AVD, HPS5 CATHL2, S100A12, EXFABP, RSFR, LY86, PKD4, XCL1 and FOS (Fig. 5B). Among these 10 DEGs, only XCL1 and FOS were significantly down-regulated in the LPS group (P < 0.05), while the remaining ones were significantly up-regulated (P < 0.05). These results indecate that the F1 hens produces a certain immune memory to the F0 LPS maternal stimulation, and the innate immunity and humoral immunity of the F1 hens are activated, which further autonomously stimulates the regulatory pathways to external stimuli. These genes could be used as genetic markers for the transmission of maternal immune stimulation between generations.
The decrease of egg-laying rate might result from the immune system deterioration of hens
It was reported that the diversity of LPS structures and the differential recognition of these structures by TLR4 had been associated with several bacterial diseases . LPS could induce innate immunity with Alzheimer’s disease  and airway disease. It also increases the invasive ability of pancreatic cancer cells through the TLR4/MyD88 signaling pathway . So the IPA was used to analyze the Diseases or Functions Annotation to study the effect of LPS maternal stimulation on the immunity of the challenged hens and their offspring. We extracted the top20 terms of both F0 and F1 generations and found that 90% (18) of the terms are shared in the two generations, and these terms were mostly involved in diseases such as cancer and tumors (Table 3, all terms are shown in Table S5 and S6), revealing that LPS could affect the health of F0 and the unchallenged F1 hens. We also found several terms related to reproduction in the F0 such as development of female reproductive tract, development of ovary, abnormal morphology of ovary and in the F1 such as development of reproductive system. Two common terms morphology of ovary and tumorigenesis of reproductive tract were also found in the two generations (Table S5 and S6). These terms indicated that the LPS maternal stimulation might negatively impact on the reproductive system of laying hens, and this influence has a succession effect, which might also explain the decline in egg-laying rate of F0 and F1 hens.
Moreover, the IPA was used to do interaction network analysis using the DEGs of F0 and F1 (Fig. 6A, B and Fig. S1), and found that the second network of F0 was related to Organismal Injury and Abnormalities (Fig. S1B) and the third network was related to Antimicrobial Response and Inflammatory Response (Fig. S1C), which indicates that maternal response to LPS caused an immune inflammatory response. The top3 networks in the F1 generation are mainly enriched in metabolism and development-related pathways, such as drug metabolism, carbohydrate metabolism, lipid metabolism and embryonic development (Fig. 6A,B and Fig. S1D). It is worth noting that we found TREM2 and MST1 in the first and third networks of the F1 generation respectively (Fig. 6A,B). Our previous studies found these two genes to be significantly negatively correlated with the egg-laying rate . At the same time, we also found TREM2 and MST1 were in the PPI network predicted with the shared DEGs of F0 and F1 (Fig. 6C), indicating the importance of these two genes in the intergenerational transmission effect of maternal stimulation. Moreover, MST1 gene is highly negatively correlated with the egg-laying rate of F1 53-week-old hens (r = -0.97, P = 0.001, Fig. 6D). The significantly up-regulation of MST1 in the LPS group of F0 and F1 generations (Fig. 6E) may be closely related to the decrease in the egg-laying rate of the two generations hens. These findings suggested that the LPS could induce subfertility and influence the immunity of the unchallenged offspring via the transmissible transcriptome. Additionally, the decline in the immunity may compromise the laying rate.
Transgenerational transmission of transcriptome induced by the LPS maternal stimulation
Although the F2 generation RNA-seq data comes from the spleen of 1-day-old chicken, the common DEGs obtained from the spleen tissues of F0, F1, and F2 chickens are still of reference value for the study of multi-generational transmission effects. The splenic transcriptome of the F2 generation was further analyzed to investigate the transgenerational transmission effect of transcriptome changes induced by the maternal stimulation. The results showed that the LPS maternal stimulus induced 2791 DEGs in the F2 generation, including 1741 up-regulated DEGs and 1050 down-regulated DEGs (Fig. 7A, P < 0.05, FC > 1.5). A total of 37 common DEGs were found among the F0, F1 and F2 generations, in which 10 DEGs have the same expression trend, including ENSGALG00000045832, ENSGALG00000036119, C3, CEP162, F13A1, GDPD2, MMR1L3, LY86, PDK4 and PPEF2 (Fig. 7B, C), revealing that the effect of maternal stimulation on the transcriptome of laying hens has a transgenerational transmission effect. The PPI networks showed that MMR1L3 and LY86 proteins are related to TLR4 (Fig. 7D, E), which is the main receptor of the LPS , indicating the transgenerational transmission effect of transcriptome may result from the LPS maternal stimulation.
Regulation of transgenerational transmission of transcriptome on egg-laying rate of hens
In addition, we extracted the FPKM of the F1 hens of the above 10 DEGs with transgenerational transmission effect and conducted a Pearson correlation analysis with the egg-laying rate of the F1 hens at 53 weeks of age. Five genes (50%) were found to have a correlation of more than 0.5 with egg-laying rate, including MMR1L3, C3, F13A1, LY86 and GDPD2 (Fig. 8). Among them, MMR1L3, C3, F13A1 and LY86 genes were negatively correlated with egg-laying rate, depicting that these genes inhibit the reproductive performance of laying hens. The GDPD2 gene was positively correlated with the egg-laying rate, indicating that this gene can moderately promote the reproductive performance of hens (Fig. 8). The significantly up-regulation of MMR1L3, C3, F13A1 and LY86 genes and the significantly down-regulation of GDPD2 in the LPS group of the three generations of hens indicate that the LPS maternal stimulation may promote the expression of MMR1L3, C3, F13A1, LY86 genes while inhibiting the expression of GDPD2 gene to reduce the egg-laying rate of hens, and this effect may has a transgenerational transmission effect.