The ACE2 expression in Sertoli cells and germ cells may cause male reproductive disorder after SARS‐CoV‐2 infection

Abstract The serious coronavirus disease‐2019 (COVID‐19) was first reported in December 2019 in Wuhan, China. COVID‐19 is an infectious disease caused by severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2). Angiotensin converting enzyme 2(ACE2) is the cellular receptor for SARS‐CoV‐2. Considering the critical roles of testicular cells for the transmission of genetic information between generations, we analyzed single‐cell RNA‐sequencing (scRNA‐seq) data of adult human testis. The mRNA expression of ACE2 was expressed in both germ cells and somatic cells. Moreover, the positive rate of ACE2 in testes of infertile men was higher than normal, which indicates that SARS‐CoV‐2 may cause reproductive disorders through pathway activated by ACE2 and the men with reproductive disorder may easily to be infected by SARS‐CoV‐2. The expression level of ACE2 was related to the age, and the mid‐aged with higher positive rate than young men testicular cells. Taken together, this research provides a biological background of the potential route for infection of SARS‐CoV‐2 and may enable rapid deciphering male‐related reproductive disorders induced by COVID‐19.

2(ACE2) is the cellular receptor for SARS-CoV-2. Considering the critical roles of testicular cells for the transmission of genetic information between generations, we analyzed single-cell RNA-sequencing (scRNA-seq) data of adult human testis. The mRNA expression of ACE2 was expressed in both germ cells and somatic cells. Moreover, the positive rate of ACE2 in testes of infertile men was higher than normal, which indicates that SARS-CoV-2 may cause reproductive disorders through pathway activated by ACE2 and the men with reproductive disorder may easily to be infected by SARS-CoV-2. The expression level of ACE2 was related to the age, and the midaged with higher positive rate than young men testicular cells. Taken together, this research provides a biological background of the potential route for infection of SARS-CoV-2 and may enable rapid deciphering male-related reproductive disorders induced by COVID-19.

K E Y W O R D S
ACE2, COVID-19, reproductive disorder, scRNA-seq, testicular cells Infertility is the most serious type of reproductive disorder, here, we performed to analyse scRNA-seq data of testis from five donors with health and eight donors with infertility. 7

| Identification of cell types and gene expression analysis
To obtain potential marker genes, Seurat function FindAllMarkers was used to per-form differential expression analysis with the default parameters. UMAP was used for visualization purposes.
Besides, Canonical markers for cell clusters were selected and plotted using ggplot2 (version 3.2.1).
According to the previous research articles, SARS-CoV-2 tends to attack cells through receptor ACE2, therefore, we defined that ACE2 positive ratio is the number of ACE2 positive cells which expression value is larger than 0 within a cluster divided by a cluster total cell number. To clarify the relationship between ACE2 and regulatory protein, we used RNA and protein network data of ACE2 through GeneCards (https://www.genec ards.org/). The relationship network data were imported into Cytoscape (version 3.7.2), and ACE2 was highlighted in yellow.

| scRNA-seq analysis of the human testicular cells
The scRNA-seq offers new possibilities to address biological and medical questions, the diverse scRNA-seq protocols and depths showed different efficient for transc-riptome quantification, so we performed to two data from different sources for analysis. 7,8 Group one with mid-aged, the different donors were labelled for more accurate data analysis with cells coloured based on their donors origin ( Figure S1A). 12 different cell types with four types of somatic cells and eight germ cell types were identified ( Figures S1B and S2).
We also analysed the data from young men for comparison, donors marked with a different colour ( Figure S1C). After initial quality controls, 12 different cell types were identified in testis (Figures S1D and S3).

| The ACE2 expression in human testicular cells
The mRNA of ACE2 were expressed in somatic and germ cells ( Figure 1A,C). In mid-aged, about 9% cells with ACE2 positive in SSCs, and Sertoli cells with higher ACE2 enrichment (90% of cells; Figure 1E). In young group, the mRNA expression of ACE2 was still identified in similar cell types ( Figure 1B

| The analysis of ACE2 between healthy and infertility men
We noticed that the tendency was similar in two groups, but the specific data were different. The mid-aged group included some pathological donors, so it was inferred that the expression of ACE2 is associated with reproductive disorders. To further de-termine the characteristic expression of ACE2, we analysed the proportion of ACE2 positive cells in different donors and found that the positive rate of the normal male was lower than that reproductive dysfunction ( Figure 2A). The testicular cells of the infected SARS were seriously damaged, IgG was highly expressed in Sertoli cells and germ cells of the patients, and there was also expressed in the Leydig cells, which was similar to the distribution of ACE2 in the testis. 9

| The mRNA expression of ACE2 in testis at different ages
SARS-CoV-2 showed a various infection rate at different ages. 10 We analysed the positive cell rate of ACE2 in testis with healthy men, the highest was 2.84% in 30-year-old donor, followed by 20-year-old donors (1.37%), and the 60-year-old donors were about 0.87% ( Figure 2B-D). Moreover, the online data of GeneCards was analysed to explore potential regulatory mechanism of ACE2 in testis ( Figure 2E). The results showed that ACE2 has related regu-

| D ISCUSS I ON
ACE2 is a functional receptor for coronaviruses. 4 In this study, we explored scRNA-seq data and found that the proportion of ACE2 positive cells in testis is more than 1%, which is higher than that in lung, indicated that the testis might serve as a high-risk potential infection organ. 6 The testicular cell type with inflammation in SARS patients is consistent with the Sertoli cell cluster in our analysis, where ACE2 expression is abundant. 9 We noticed that the positive rate of ACE2 in testis of infertile was higher than normal men, which indicates that SARS-CoV-2 may cause reproductive disorders through abnormal activation of ACE2 pathway. One study found that SARS-CoV-2 in the semen of some male patients with COVID-19, further implicating that testis is a portal of infection. 12 In addition, ACE2 is widely distributed in SSC, Sertoli cells, and Leydig cells. These findings indicate these cells as possible loci of infection and sexual transmission might be a part of the transmission routes.
SARS-CoV-2 had various infection rates in humans at different ages, and men had a higher prevalence than women. 13 Previous studies have shown that gender was an important factor affecting ACE2 concentrations in plasma. 13 Therefore, we investi-gated the positive rate of ACE2 in testicular cells of man at different ages. Here, we found that 30-year-old men with the highest ACE2 positive rate and 60-year-old men with the lowest ratio in testis.
Men had higher mortality than women in the SARS epidemic. 14 Moreover, SARS-CoV had a lower lethality rate for 2-month-old male mice than 8-month-old male mice and relatively higher mortality in middle-aged mice. 15 These data suggest that viral infection might be associated with androgen secretion. Different treatment regimens should be appropriate for male and female patients considering androgen levels. Our studies may help with COVID-19 prevention and control.

Program of Shaanxi Province Science and Technology Innovation
Team (2019TD-036).

CO N FLI C T O F I NTE R E S T
The authors of this manuscript have no conflicts of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.