Co-expression of the SARS-CoV-2 entry molecules ACE2 and TMPRSS2 in human ovaries: Identification of cell types and trends with age

Meng Wu, Lingwei Ma, Liru Xue, Qingqing Zhu, Su Zhou, Jun Dai, Wei Yan, Jinjin Zhang, Shixuan Wang, Meng Wu, Lingwei Ma, Liru Xue, Qingqing Zhu, Su Zhou, Jun Dai, Wei Yan, Jinjin Zhang, Shixuan Wang

Abstract

The high rate of SARS-CoV-2 infection poses a serious threat to public health. Previous studies have suggested that SARS-CoV-2 can infect human ovary, the core organ of the female reproductive system. However, it remains unclear which type of ovarian cells are easily infected by SARS-CoV-2 and whether ovarian infectivity differs from puberty to menopause. In this study, public datasets containing bulk and single-cell RNA-Seq data derived from ovarian tissues were analyzed to demonstrate the mRNA expression and protein distribution of the two key entry receptors for SARS-CoV-2-angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2). Furthermore, an immunohistochemical study of ACE2 and TMPRSS2 in human ovaries of different ages was conducted. Differentially expressed gene (DEG) analysis of ovaries of different ages and with varying ovarian reserves was conducted to explore the potential functions of ACE2 and TMPRSS2 in the ovary. The analysis of the public datasets indicated that the co-expression of ACE2 and TMPRSS2 was observed mostly in oocytes and partially in granulosa cells. However, no marked difference was observed in ACE2 or TMPRSS2 expression between young and old ovaries and ovaries with low and high reserves. Correspondingly, ACE2 and TMPRSS2 were detected in the human ovarian cortex and medulla, especially in oocytes of different stages, with no observed variations in their expression level in ovaries of different ages, which was consistent with the results of bioinformatic analyses. Remarkably, DEG analysis showed that a series of viral infection-related pathways were more enriched in ACE2-positive ovarian cells than in ACE2-negative ovarian cells, suggesting that SARS-CoV-2 may potentially target specific ovarian cells and affect ovarian function. However, further fundamental and clinical research is still needed to monitor the process of SARS-CoV-2 entry into ovarian cells and the long-term effects of SARS-CoV-2 infection on the ovarian function in recovered females.

Keywords: ACE2; Ovary; SARS-CoV-2; Single-cell RNA-Seq; TMPRSS2.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Copyright © 2021. Published by Elsevier Inc.

Figures

Fig. 1
Fig. 1
mRNA expression levels of ACE2 (A) and TMPRSS2 (B) in multiple organs; data obtained from the Genotype-Tissue Expression (GTEx) database (a human organ database); * p < 0.05, ** p < 0.001, *** p = 0.0001, **** p < 0.0001. The vertical coordinates represent the log transformed (log2 [x + 0.001]) relative expression level of ACE2 and TMPRSS2.
Fig. 2
Fig. 2
Gene expression profiles of distinct ovarian cell subpopulations based on ovarian scRNA-Seq data from dataset GSE130664 (macaque ovarian single cells). (A) T-distributed stochastic neighbor embedding (t-SNE) clustering of adult ovarian cells into eight clusters based on the scaled data. (B) Average expression level of ACE2 (upper) and TMPRSS2 (lower) in each cell cluster. The vertical coordinates represent the log-transformed [log (x + 1)] relative expression level of ACE2 and TMPRSS2. (C) Scatter plot of ACE2 and TMPRSS2 gene expression of each cell cluster. The ellipse indicates the cell cluster of oocytes that co-expressed ACE2 and TMPRSS2. The number and gradient color of the legend bars show the log-transformed values of gene expression matrix raw data of each cell cluster. (D) Dot plot of the well-known markers including ACE2 and TMPRSS2 in each cell cluster. The color of the legend bar of average expression reflects the mean expression. The size of the dots corresponds to the percentage of cells expressing the listed genes in each cell cluster. (E) Scatter plots of cell clusters with canonical markers of oocytes and (F) granulosa cells.
Fig. 3
Fig. 3
Gene expression profiles of oocytes and granulosa cells based on the ovarian scRNA-Seq data from dataset GSE107746 (human oocytes and granulosa cells). (A) T-distributed stochastic neighbor embedding (t-SNE) clustering of adult ovarian cells into two clusters—oocytes and granulosa cells. (B) Average expression level of ACE2 (upper) and scatter plot of ACE2 expression (lower) in each cell cluster based on the canonical cell markers of oocytes and granulosa cells. GCs: granulosa cells. The number and gradient color of the legend bars showed the log-transformed values of gene expression matrix raw data of each cell cluster. (C) Average expression level of TMPRSS2 (upper) and the scatter plot of TMPRSS2 expression (lower) of each cell cluster. (D) Dot plot of the well-known markers including ACE2 and TMPRSS2 in each cell cluster. The color of the legend bar of average expression reflects the mean expression. The size of the dots corresponds to the percentage of cells expressing the listed genes in each cell cluster. (E) Scatter plots of cell clusters with the markers of oocytes and (F) granulosa cells.
Fig. 4
Fig. 4
ACE2 or TMPRSS2 gene expression did not significantly differ between old and young ovaries. (A) Dot plot of each cell cluster in GSE130664 (macaque ovarian single cells) in old and young groups, showing the expression level and percentage of cells in a cluster expressing conserved markers (including ACE2 and TMPRSS2). (B) Comparative analysis of oocytes and granulosa cells in GSE130664 (macaque ovarian single cells) to visualize ACE2 and TMPRSS2 expression with the outlier scatter plot. The ellipse indicates the differentially expressed genes between old and young ovarian single cells. (C) Violin plots show the expression level and distribution of ACE2 and TMPRSS2 in all ovarian single cells from all seven cell types of each ovarian cell cluster in old and young groups in GSE130664 (macaque ovarian single cells). The vertical coordinates represent the log-transformed [log (x + 1)] relative expression level of ACE2. The average expression level of ACE2 and TMPRSS2 showed no significant differences between old and young ovarian single cells. (D) Volcano plot of aged and young human cumulus cells in dataset GSE81579 (bulk RNA-Seq of human cumulus cells), with ACE2 (black circle) included among the genes that showed non-significant differential expression (grey circles); however, TMPRSS2 was not detected. (E) Volcano plot of bulk RNA-Seq of aged and young ovarian tissues in Genotype-Tissue Expression (GTEx) project (bulk RNA-Seq of human ovarian tissues); neither ACE2 nor TMPRSS2 (black circles) was differentially expressed. (F) Volcano plot of oocytes with low and high ovarian reserve in dataset GSE87201 (bulk RNA-Seq of human oocytes), neither ACE2 nor TMPRSS2 (black circles) was differentially expressed.
Fig. 5
Fig. 5
Representative images of ACE2 (A) and TMPRSS2 (B) immunohistochemical staining in the human ovarian cortex of different ages (14 to 60 yr). The positive control was human testis. The negative control was carried out by omitting the primary antibody.
Fig. 6
Fig. 6
Immunohistochemical staining indicated that the expression of ACE2 (A) and TMPRSS2 (B) occurred in all types of human follicles. The red arrows indicate follicles. The negative control was carried out by omitting the primary antibody. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 7
Fig. 7
Immunofluorescence staining showed the co-expression of ACE2 and TMPRSS2 in human ovaries. The yellow arrows indicate follicles. The blue channel is DAPI staining, the green channel is ACE2 expression, and the red channel is TMPRSS2 expression. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 8
Fig. 8
Differentially expressed gene (DEG) functional analysis of ACE2 and TMPRSS2 in macaque single ovarian cells (GSE130664). (A) Gene ontology (GO) enrichment results (only biological process terms shown) of DEGs in ACE2-positive and ACE2-negative ovarian cells. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs in ACE2-positive and ACE2-negative ovarian cells. (C) Visualization of the relationships of GO terms and corresponding mapped genes. (D) GO enrichment result (only BP terms shown) of DEGs in TMPRSS2-positive and TMPRSS2-negative ovarian cells. (E) KEGG pathway enrichment analysis of DEGs in TMPRSS2-positive and TMPRSS2-negative ovarian cells. BP: biological process.
The following are the supplementary data related…
The following are the supplementary data related to this article.Supplementary Fig. 1
The expression profile of BSG and CTSL in various datasets. (A) Feature plot of BSG expression in all ovarian cells in GSE130664 (macaque ovarian single cells). The legend bars represent a log-transformed gene expression value. (B) Dot plot of BSG, TMPRSS2, and ACE2 expression for each major cell population in GSE130664. (C) Feature plot of BSG and CTSL expression in all cells in GSE107746 (human oocytes and granulosa cells). (D) Dot plot of BSG, CTSL, TMPRSS2, and ACE2 for the two subpopulations in GSE100746.
Supplementary Fig. 2
Supplementary Fig. 2
Representative images of ACE2 (A) and TMPRSS2 (B) immunohistochemical staining in the medulla of human ovaries of different ages.
Supplementary Fig. 3
Supplementary Fig. 3
Quantification of immunofluorescence intensity of expression for both ACE2 and TMPRSS2.

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