COVID-19 disrupts spermatogenesis through the oxidative stress pathway following induction of apoptosis

Abstract

To evaluate the incidence of apoptosis within the testes of patients who died from severe acute respiratory syndrome coronavirus 2 (COVID-19) complications, testis tissue was collected from autopsies of COVID-19 positive (n = 6) and negative men (n = 6). They were then taken for histopathological experiments, and RNA extraction, to examine the expression of angiotensin-converting enzyme 2 (ACE2), transmembrane protease, serine 2 (TMPRSS2), BAX, BCL2 and Caspase3 genes. Reactive oxygen species (ROS) production and glutathione disulfide (GSH) activity were also thoroughly examined. Autopsied testicular specimens of COVID-19 showed that COVID-19 infection significantly decreased the seminiferous tubule length, interstitial tissue and seminiferous tubule volume, as well as the number of testicular cells. An analysis of the results showed that the Johnsen expressed a reduction in the COVID-19 group when compared to the control group. Our data showed that the expression of ACE2, BAX and Caspase3 were remarkably increased as well as a decrease in the expression of BCL2 in COVID-19 cases. Although, no significant difference was found for TMPRSS2. Furthermore, the results signified an increase in the formation of ROS and suppression of the GSH activity as oxidative stress biomarkers. The results of immunohistochemistry and TUNEL assay showed that the expression of ACE2 and the number of apoptotic cells significantly increased in the COVID-19 group. Overall, this study suggests that COVID-19 infection causes spermatogenesis disruption, probably through the oxidative stress pathway and subsequently induces apoptosis.

Keywords: Apoptosis; COVID-19; Spermatogenesis disrupt; Testicular tissue.

Fig. 1

(I) Chest X-ray image of a COVID-19 positive old male, showing bilateral mid and lower zones homogenous consolidation in peripheral distribution along with Obscuration of both CP angles (red arrow), findings fall in the category of indeterminate for COVID-19 (A–F). (II) Axial thin-section chest computed tomography (CT) in patients with COVID-19 pneumonia from our institution. A–G Non-contrast chest CT scan exam showed bilateral ground glass opacities and interlobular septal thickening giving appearance of crazy paving and areas of surrounding consolidation giving appearance of reverse halo sign and air bronchogram (red arrow) (Color figure online)

Fig. 2

A In the control group, the lung parenchyma, including the alveolar sac and alveolar walls, has a normal appearance. B In Covid-19 group, infiltration of lung tissue by mononuclear inflammatory cells (arrow), along with desquamation of alveolar epithelium and formation of hyaline membrane together alveolar wall thickening was observed

Fig. 3

Photomicrograph of the testis stained with H&E; (× 4, × 10 and × 40). SG Spermatogonia (SG), primary spermatocyte (PS), round spermatid (ST), Sertoli cell (SC), Leydig cell (LC). A In the control group, the testis tissue, including the seminiferous tubules and germinal epithelium, has a normal appearance. B The testicular cells death, interstitial edema and thinning of the seminiferous epithelium was observed in all autopsy COVID-19 specimens of testes. C A point grid is superimposed over the photomicrograph for measuring the volume using Cavalieri method. D Counting frames are superimposed over the photomicrographs to measure the number of testicular cells using optical disector method. The yellow box is counting frame (Color figure online)

Fig. 4

A–C Stereological results of testis tissue. Mean ± SD of the seminiferous tubules length, interstitial tissue volume and seminiferous tubules volume of testis in the study groups; (***P < 0.001). C Photomicrograph of the testis stained with H&E, × 4. D–H Stereological results of testis tissue. Mean ± SD of the total number of spermatogonia, total number of primary spermatocyte, total number of round spermatid, total number of Leydig cells and total number of Sertoli cells in the study groups; (***P < 0.001)

Fig. 5

Photomicrograph of the testis stained with H&E; (× 40). A Histopathological finding showed normal spermatogenesis in all control autopsy and the Johnsen score was 10. B Testicular histopathology in the six COVID-19 autopsy specimens such as: sample (I) had some tubules with slightly impaired spermatogenesis and the Johnson score was 9. Two samples (II and III) had some tubules with less than five spermatozoa per tubule and the Johnson score was 8. samples (IV and V) had some tubules with no late spermatids and many early spermatids and the Johnson score was 7. Sample (IV) had some tubules with few early spermatids; many of spermatogenesis at the spermatid stage and the Johnson score was 6. C Mean ± SD of the Johnsen score in the study groups; (*P < 0.001)

Fig. 6

Photomicrograph of the testis immunohistochemistry staining for ACE2 (4X and 40X). A Control group and B COVID-19 groups. Expressed of ACE2 in Leydig cells (arrows) according to immunohistochemistry. C Mean ± SD of the genes expression in the different groups (***P < 0.001)

Fig. 7

A ROS production and level of GSH in testis tissue. Mean ± SD of the ROS production and GPX activity level in the study groups; (*P < 0.05). B Real-time-PCR analyses. mRNA expression levels of (ACE2, TMPRSS2, BAX, BCL2 and Caspase3) in the testis tissue. Mean ± SD of the genes expression in the different groups (*P < 0.05 and ***P < 0.001). PCR, polymerase chain reaction

Fig. 8

The percent of apoptotic cells. Tunel detection of apoptotic cells in the testes of the A Cont group and B COVID-19 group. TUNEL positive cells with brown neuleous (Arrows). C Mean ± SD of the percent of apoptotic cells of testis in control and COVID-19 groups. (***P < 0.001) (Color figure online)