## 4 Discussion
Previous reports show that the number of Sertoli cells determines the size of the testis and the number of spermatozoa produced [44]; consequently, a loss of Sertoli cells is associated with diminished spermatozoa production [44; 48; 49]. Furthermore, consistent with our results, diabetes has been reported to cause Sertoli cell degeneration [50; 51]. Alcohol has deleterious effects on Sertoli and Leydig cells [52; 53], which may substantiate the observed toxicity. Additionally, the results show significant decreases in the Leydig cell nuclear diameter (LCND) and volume (LCNV) in all treated groups, indicating a possible detrimental effect of treatment on the steroidogenic capacity, since studies have shown that alterations in the nuclear volume and size are functionally associated [54]. In agreement with our findings, [PERSON] et al. (2015) [55] observed necrotic Leydig cells in the testes of mice treated with alcohol, and [PERSON] et al. (2018) [27] reported reduced LCNVs in rats treated with cART. Further, [PERSON] et al. (2012) [56] found changes in the ultrastructures of Sertoli and Leydig cells in the testes of diabetic male rats. Conversely, via the steroidogenesis process, Leydig cells produce testosterone, a male androgen that is essential for successful spermatogenesis. The Leydig cell steroidogenic capacity is suggested to be determined by the number of cell nuclear organelles, such as mitochondria, rather than the number of cells themselves [43; 57]. Previously, researchers have shown that a reduction in the Leydig cell nuclear volume is associated with a decreased testosterone level [57; 58]. Thus, the observed deleterious alternations of the Sertoli and Leydig cells induced by the treatment may elicit the dysfunction of these testicular somatic cells, which will eventually lead to spermatogenesis impairments. While the cellular components of the testes are crucial for successful reproduction, the surrounding testicular capsule and the stroma, which is sandwiched between the semiiferous tubules, also play essential roles. These connective tissue components are essential for the maintenance of the testicular structure and the germinal epithelium integrity [59; 60]. Recently, capsular, interstitial, and peritubular connective tissues have also been recognized to play roles in the transportation of spermatozoa, particularly due to the contractility of myoid cells, which aids in the propulsion of spermatozoa from the tubule lumen to the retets testis [33]. Therefore, the observed significant increase in the thickness of the semiiferous tubule basement membrane (STBM) in the testes of the DM+A and DM+cART groups and the testicular capsule in the DM+A and DM+A+cART groups suggest structural distortion induced by treatments which may adversely affect their respective functions. In agreement with our findings, [PERSON] et al. (2021) [61] found increases in the STBM and testicular capsule thickness in diabetic rats, and in the present study, such increases were observed in the combinations of DM+cART and DM+A+cART. Further, a distorted capsular collagen orientation has been reported following alcohol administration [62]. Our results further show that connective tissue fibers (collagen, reticulum, and elastin) were reduced in treated groups relative to the control group, which is consistent with previous reports that documented reductions in collagen and reticul fibers in rats treated with alcohol [63; 64]. Similarly, a loss of elastin fibers has been reported in the skin and vasculature of diabetic individuals [65; 66]. The alternations observed in testicular connective tissue show that the treatments disintegrated the structural integrity of the testes, subsequently leading to testicular dysfunction. Furthermore, the testicular structure and functions are regulated by hormones of the hypothalamic-pituitary-gonadal (HPG) axis [43; 67]. Accordingly, changes in reproductive hormone levels will lead to the dysregulation of steroidogenesis and spermatogenesis.