Impact of Concurrent Exposure of Diabetic Male Sprague Dawley Rats to Alcohol and Combination Antiretroviral Therapy (cART) on Reproductive Capacity

Abstract

The prevalence of diabetic patients who abuse alcohol while on combination antiretroviral drug therapy (cART) therapy is rising in society. Little is known about the impact of this scenario on the testes and male reproductive viability, and therefore, these factors were evaluated. Thirty 10-week-old male Sprague Dawley rats were distributed into five groups of six rats each: control, diabetic only (DM), diabetic treated with alcohol (DM+A), diabetic treated with Atripla, fixed-dose cART (DM+cART), and diabetic treated with both alcohol and cART (DM+A+cART). After 90 days of treatment, rats were terminated, and blood and testes were harvested for immunoassay, histological, and immunohistochemistry analyses. Testicular perturbations of varying severity were recorded in all treated groups for most of the parameters. The DM+A treated group showed the most severe perturbations, followed sequentially by the treated groups DM+A+cART, DM, and DM+cART. Alterations in the testes and seminiferous tubule morphometry as well as the spermatogenic, Sertoli, and Leydig cells were found in all treated groups. Further, a significant decrease in Johnsen’s testicular scores, the appearance of seminiferous tubule lesions, changes in the basement membrane and capsule thickness, and a reduction in the testis connective tissue fibers were demonstrated in the treated groups. Additionally, reproductive hormone levels were altered, and the number and staining intensity of Sertoli and Leydig cells expressing androgen receptors reduced significantly in all treated animals. The study results reveal that the consumption of alcohol and/or the use of cART in diabetic individuals induces a derangement in circulating reproductive hormone levels and in the testicular structure and function, which consequently leads to a decline in the male reproductive capacity.

1. Introduction

The decline in male fertility is increasingly becoming a global concern [1] and is associated with rising incidences of obesity, drug abuse, noncommunicable diseases (NCDs), and environmental toxins [2,3]. Diabetes mellitus (DM) is a chronic disorder of glucose dysregulation, marked by persistent high blood glucose levels (hyperglycemia) [4]. DM is one of the most common NCDs globally and has emerged as a public health burden in sub-Saharan Africa [5]. In South Africa, the prevalence of DM has almost doubled since 2017, from 5.4% to the current 11.3% [4]. The fast-rising incidence of DM has been linked to the predominance of high carbohydrate staple foods, obesity, and excessive alcohol consumption in South Africa [5]. Additionally, more than 33% of the South African population consumes alcohol regularly [6]. Unfortunately, South Africa is also faced with a high prevalence of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), with almost 20% of the adult population living with HIV/AIDS, warranting extensive use of combination antiretroviral therapy (cART) [7].

Incidentally, an increased number of males within reproductive age are impacted by diabetes [8], alcohol abuse [9], and the cART regimen [2]. Moreover, an intricate connection exists among these conditions; for instance, alcohol is a recognized risk factor for diabetes [10] and has also been implicated in increased HIV spread and consequently is linked to the rise in cART use [11]. Equally, an increase in alcohol use in individuals living with HIV, including those on cART, has also been reported [12]. Further, both HIV infection and cART have been implicated in diabetes induction [13]. These three conditions can simultaneously occur in an individual; thus, the associated testicular histopathology and reproductive health implications need to be documented.

Moreover, diabetes, alcohol consumption, and cART use have each been reported to affect the male reproductive system. Studies on diabetes have reported reproductive hormone imbalances, testicular structure derangements, seminiferous tubule lesions, a reduced ejaculate volume, poor sperm quality, and sexual dysfunctions in diabetic individuals [8,14], as well as in those who abuse alcohol [15,16,17] and in cART users [18,19,20]. Unfortunately, there is no scientific literature available on the impact of the multimorbidity of these conditions (diabetes, alcohol abuse, and cART) on male fertility. However, such studies are particularly relevant, since these conditions exist in society and are particularly rampant in the population of reproductive age. Hence, this study reports on the impact of the co-presence of alcohol and cART in diabetic individuals on the testicular histomorphology as well as possible consequences for the reproductive capacity.

2. Materials and Methods

2.1. Chemical and Reagents

Streptozotocin (STZ) (S0130) was purchased from Sigma–Aldrich Chemical Company (St. Louis, MO, USA) and Atripla, a fixed-dose combination antiretroviral drug, was purchased from Bristol–Myers Squibb and Gilead Sciences (Foster City, CA, USA). Androgen receptor rabbit monoclonal (ab105225) primary antibody was procured from Abcam (Cambridge, MA, USA), and biotinylated goat antirabbit (BA-1000) secondary antibody and the Avidin–Biotin Complex kit (ABC) (PK-6100) were procured from Vector Laboratories (Burlingame, CA, USA). Then, ELISA kits for luteinizing hormone (LH) (E-EL-R0026), follicle-stimulating hormone (FSH) (E-EL-R0391), testosterone (TT) (E-EL-R0155), and inhibin B (INHB) (E-EL-R1027) were procured from Elabscience (Houston, TX, USA).

2.2. Ethical Clearance

The Animal Research Ethics committee (AREC) of the University of the Witwatersrand (Wits) approved this study (approval number: 2018/011/58/C), and all experiments were carried out at the Wits Research Animal Facility (WARF) following AREC guidelines.

2.3. Animal Husbandry

Thirty (30) adult (10-week-old) male Sprague Dawley rats were utilized. Rats were kept apart in a sterile plastic cage and housed at room temperature (21–23 °C) with a 12/12-h light/dark cycle, fed with standard rat chow, and given drinking water ad libitum.

2.4. Type 2 Diabetes Induction

A modified procedure described by Wilson and Islam in 2012 [21] was used to induce type 2 diabetes. Briefly, animals were fed a 20% fructose reconstituted rat chow diet for two weeks, after which a single injection of 40 mg/kg streptozotocin (STZ) in 0.05 M (pH 4.5) citrate buffer (freshly prepared) was administered intraperitoneally. Blood glucose (nonfasting) levels were measured 72 h after STZ administration, and rats with glucose levels ≥ 250 mg/dL were regarded as diabetic. Once the diabetic state was confirmed in animals, they were placed on a standard rat chow diet. Further, fasting blood glucose (FBG) levels were measured biweekly following a 12-h fasting period (20:00 to 08:00) through a tail prick using the Accu-Chek meter and glucose strips.

2.5. Experimental Design

The animals were divided into five groups, each with six animals, as follows: control group, diabetic (DM) group, diabetic animals treated with 10% v/v alcohol daily (DM+A), diabetic animals treated with an extrapolated human equivalent dose of 23 mg/kg of cART (consisting of efavirenz 600 mg + emtricitabine 200 mg + tenofovir 245 mg) [22] in gelatine cubes daily (DM+cART), and diabetic animals treated with both alcohol and cART (DM+A+cART). The animals were treated for 90 days concurrently, after which the animals were weighed, anesthetized with 240 mg/mL pentobarbitone, and terminated. Blood was withdrawn through a cardiac puncture into a plain vacutainer. Then, animals were perfused with 2 mL/min of 0.1 M phosphate buffer (PB) in 0.9% saline before extraction of the testes. The testes were then preserved in 10% neutral buffered formalin for subsequent processing. Collected blood samples were left to clot, then centrifuged, and the serum was transferred into a new Eppendorf tube and stored at −80 °C for immunoassays.

2.6. Testis Morphometry

2.6.1. Testis Weight and Volume

The weight of each testis was recorded immediately after extraction using an electronic scale (Kern PCB 200-2 weighing scale). The testis volume was determined using the Archimedes principle of water displacement in a measuring cylinder [23]. The initial volume (Vi) of distilled water in a graduated cylinder was recorded, and then the final volume (Vf) was determined after adding the testis to the graduated cylinder containing a known amount of distilled water. The volume of the testis (Vt) was calculated by subtracting the initial volume (Vi) from the final volume (Vf), as shown below.

$$Vt=Vf-Vi \left(\mathrm{m}{\mathrm{m}}^3\right)$$

2.6.2. Testis Size

A sliding digital Vernier caliper was used to measure the width and length of each testis, as described by Parhizkar et al., 2014 [24]. The testis size was then calculated using the spheroid formula:

$$Testis size=widt{h}^2\times length\times 0.523 \left(\mathrm{m}\mathrm{m}\right)$$

2.7. Histology and Histomorphometry (H&E)

The fixed testes were dehydrated sequentially in 70–100% alcohol grades and embedded in molten paraffin wax and sectioned at 5-μm thicknesses. The sections were stained with H&E and examined using the Axioskop 2 plus light microscope (Nikon Eclipse Ci, 104C type). Photomicrographs of testicular tissue sections were obtained using a linked computerized Zeiss digital image system, the AxioCam 208 color (Zeiss group, Oberkochen, Germany), for morphometric assessment.

2.8. Testicular Component Area Fractions

Testicular component (seminiferous epithelium, connective tissue, lumen, and interstitial space) area fractions were determined using the Fiji 84-intersection grid. The grid was superimposed on H&E-stained images captured at ×100 magnification, and the intersecting points for each component were counted. The average number of intersecting points for each testicular component was calculated in 24 camera fields for each animal (i.e., 144 fields per group), and each testicular component area fraction was determined using the formula below [25]:

$$Area fraction=\frac{\left(area per point \times average number of intersecting points\right)}{total area of photomicrograph}\times 100 (\%)$$

2.9. Seminiferous Tubule Morphometry

The seminiferous tubule area, tubule diameter, and epithelium height were measured in 50 round or nearly round seminiferous tubules for each animal (i.e., 300 tubules per group) using Fiji software. The seminiferous tubule cross-sectional area was determined by tracing around the seminiferous tubule using the Fiji freehand selection tool. The average tubule diameter and epithelial height were calculated with the minor and major axes measurements [26]. To exclude longitudinal tubules, an average tubule diameter was considered only if D1/D2 ≥ 0.85; a value of D1/D2 = 1.0 represented a perfectly round circle [27].

2.10. Germinal Epithelium Cell Quantification

The numbers of the different germinal epithelium cells (Sertoli, spermatogonia, spermatocytes, round, and elongated spermatids) were counted in H&E-stained sections at ×400. For each animal, 10 rounded VII seminiferous tubules were considered (i.e., 60 tubules per group) [28].

2.11. Leydig Cell Morphometry

The diameters of 50 nuclei with distinct nucleoli were considered for each animal (i.e., 300 per group) and measured using Fiji software for each group on H&E-stained images captured at ×400. The nuclear volume was obtained using the formula below [29]:

$$Leydig cell nuclear volume=\frac{4}{3}\pi R^3 (\mathsf{\mu }{\mathrm{m}}^3).$$

where Radius (R) = D/2 and $\pi$ = 3.14.

2.12. Histopathological Evaluation

2.12.1. Johnsen’s Testicular Score

Spermatogenesis was classified in 50 seminiferous tubules (stages II–VII) for each animal (i.e., 300 tubules per group) using Johnsen’s testicular score. Each tubule was assigned a score from 10 to 1 according to the modified Johnsen scoring criteria [30], as follows: tubules with many spermatozoa in the tubule lumen, 10; tubules with many elongated spermatids in the apical region of the epithelium, 9; tubules with many elongated spermatids still embedded in the Sertoli cell membrane, 8; tubules with few elongated spermatids but many round spermatids, 7; tubules with no elongated spermatids but a few round spermatids, 6; tubules with no spermatids but many spermatocytes, 5; tubules with few spermatocytes, 4; tubules with spermatogonia cells only, 3; tubules with Sertoli cells only, 2; and tubules with no seminiferous epithelial cells, 1. Thereafter, the number of tubules with an individual score was multiplied by the score, and then the total for all scores was divided by 50 (the total number of tubules scored) to obtain the mean Johnsen’s score per group for further analysis [30].

2.12.2. Histological Changes

Testicular H&E-stained sections were examined for the presence of histological changes. The observed changes were then graded in 24 microscopic fields for each animal (i.e., 144 fields per group). The changes in each field were semiquantitatively categorized into five grades, as follows: grade 4, very severe (change seen in >75% of the field); grade 3, severe (change seen in >50% to ˂75% of the field); grade 2, moderate (change seen in >25% to ˂50% of the field); grade 1, mild (change seen in ˂25% of the field); and grade 0, none (no change in the field) [31].

2.13. Measurement of the Seminiferous Tubule Basement Membrane (STBM)

Photomicrographs of PAS-stained sections were captured at ×400 magnification, and Fiji software was used to measure the thickness of the basement membrane. The thickness of the STBM was measured at two points (from the minor and major axes) in 50 randomly selected tubules for each animal (i.e., 300 tubules per group) [32].

2.14. Evaluation of the Testicular Capsule Thickness

The capsule thickness was measured on Masson’s Trichrome stained sections. Photomicrographs of six fields from the free surface of the capsule were captured at ×400 magnification, and three points were measured on each field (i.e., 18 measurements for each animal, 108 per group) [33].

2.15. Interstitial Connective Tissue Quantification

Components of testicular connective tissue, i.e., collagen, reticulin, and elastin fibers, were quantified in the photomicrographs of sections stained with Masson’s Trichrome, Gordon and Sweet’s silver impregnation, and Gomori’s Aldehyde Fuchsin techniques, respectively. Collagen, reticulin, and elastin fibers were quantified in the 24 microscopic fields captured at ×100 magnification for each animal (i.e., 144 sections per group). Previously reported steps were followed to segment connective tissue images using Ilastik (v1.3.3; https://www.ilastik.org, accessed on 20 April 2021), and subsequently, the segments were quantified with Fiji software (v1.52e; https://imagej.net/Fiji, accessed on 9 October 2021) [34].

2.16. Immunoassay for Reproductive Hormones

Serum levels of the reproductive hormones, i.e., luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (TT), and inhibin B (INHB), were quantified using enzyme-linked immunosorbent assay (ELISA) in accordance with the manufacturer’s protocols. The standards and samples were run in duplicate, and the concentrations of serum TT, LH, FSH, and INHB were determined from respective standard curves plotted with the standard concentrations and their optical densities.

2.17. Immunocytochemistry (IHC) Technique for the Androgen Receptor

Testicular tissue sections of 5-μm thickness were mounted onto saline-coated slides and immunolabeled with AR antibody by following the IHC steps previously reported by us [34]. Briefly, sections were incubated with 1:100 antiandrogen receptor at 4 °C overnight. The immunoreaction was detected using 3,3′-diaminobenzidine tetrachloride (DAB) and counterstained in hematoxylin. The number of Sertoli cells expressing androgen receptor immunoreactivity was counted in 20 rounded seminiferous tubules (stage II–VII) [35] for each animal (i.e., 120 tubules for each group). The number of Leydig cells expressing androgen receptor immunoreactivity was counted in 20 microscopic fields at ×400 magnification for each animal (i.e., 120 fields for each group). Further, the staining intensity was quantified in 25 androgen receptor immunopositive Sertoli and Leydig cells for each animal group.

2.18. Data Analysis

GraphPad Prism 6 Windows software was used for the data analysis. Data were expressed as the mean ± SEM. Group means of parametric data were compared using a one-way analysis of variance, followed by Bonferroni’s post hoc test. Nonparametric data (Johnsen’s and histological change scores) were compared using the Kruskal–Wallis test, followed by Dunn’s post hoc test. p < 0.05 was considered statistically significant.

3. Results

3.1. Blood Glucose Level

Fasting blood glucose (FBG) levels increased significantly in all treated groups (DM (p = 0.0325), DM+A (p = 0.0020), DM+cART (p < 0.0001), and DM+A+cART (p = 0.0202)) relative to the control group. Further, the FBG level of the DM+cART group was significantly increased (p = 0.0499) relative to the DM group (

Table 1. Mean ± SEM fasting glucose, testis morphometry, and component area fractions of the animal groups.

Animal Groups	Control	DM	DM+A	DM+cART	DM+A+cART
Fasting glucose (mmol/L) Testis morphometry	4.99 ± 0.11	11.35 ± 1.70 a	13.97 ± 1.36 a	17.65 ± 2.31 ab	12.06 ± 0.82 a
Testis weight (g)	1.91 ± 0.05	1.69 ± 0.07	1.31 ± 0.27 a	1.72 ± 0.05	1.72 ± 0.09
Testis volume (mm3)	1.88 ± 0.09	1.63 ± 0.13	1.2 ± 0.2 a	1.67 ± 0.08	1.7 ± 0.12
Testis size (mm)	1493 ± 41	1199 ± 85	991 ± 96 a	1271 ± 49	1218 ± 103
Testicular component area fractions (AF) (%)
Epithelial (EAF)	74.25 ± 0.5	71.62 ± 0.73 ae	69.69 ± 0.58 ae	70.9 ± 0.57 ae	74.08 ± 0.42
Lumen (LAF)	8.56 ± 0.29	7.02 ± 0.23 ac	8.68 ± 0.34	7.36 ± 0.29 ac	7.59 ± 0.22
Interstitial space (ISAF)	4.93 ± 0.12	6.13 ± 0.14 a	6.12 ± 0.16 a	5.59 ± 0.15 a	5.76 ± 0.16 a
Connective tissue (CTAF)	11.97 ± 0.31	14.69 ± 0.37 a	15.12 ± 0.32 ae	15.77 ± 0.39 ae	13.53 ± 0.35 a

In the same row, different superscripts ^a, ^b, ^c, and ^e represent significant differences (p < 0.05) as analyzed by the Bonferroni’s multiple comparison test: ^a significantly different compared to the control group, ^b significantly different compared to DM, ^c significantly different compared to DM+A, and ^e significantly different compared to DM+A+cART. Key: DM, diabetes; DM+A, diabetes and alcohol; DM+cART, diabetes and combination antiretroviral therapy; DM+A+cART, diabetes and alcohol and combination antiretroviral therapy.

).

3.2. Testis Morphometry

General decreases in the testicular weight, volume, and size of all treated groups were observed relative to the control group, but only the DM+A treated group had significant decreases in weight (p = 0.0242), volume (p = 0.0096), and size (p = 0.0018) relative to the control group (Table 1).

3.3. Testicular Component Area Fractions

The epithelial area fraction (EAF) of the DM, DM+A, and DM+cART treated groups was significantly reduced relative to the control group (p = 0.0116, p < 0.0001, and p = 0.0004) and DM+A+cART (p = 0.0233, p< 0.0001, and p = 0.0009), respectively (Table 1). However, the DM+A+cART treated group showed an insignificant reduction (p > 0.05) in the EAF relative to the control group. The DM and DM+cART treated groups showed significant decreases in the lumen area fraction (LAF) relative to the control group (p = 0.0011 and p = 0.0239) and the DM+A group (p = 0.0003 and p = 0.0088). Relative to the control group, the interstitial space area fraction (ISAF) increased significantly in the DM (p < 0.0001), DM+A (p < 0.0001), DM+cART (p = 0.0196), and DM+A+cART (p = 0.0009) treated groups. A significant increase in the connective tissue area fraction (CTAF) was detected in all treated groups relative to the control group (DM (p < 0.0001), DM+A (p < 0.0001), DM+cART (p < 0.0001), and DM+A+cART (p = 0.0162)). Further, the CTAF of the DM+A and DM+cART treated groups was significantly increased relative to the DM+A+cART group (p = 0.0129 and p < 0.0001, respectively) (Table 1).

3.4. Seminiferous Tubule Morphometry

The tubule area and diameter of the DM, DM+A, and DM+cART treated groups reduced significantly (p < 0.0001) relative to the control and DM+A+cART groups (

Table 2. Mean ± SEM tubule morphometry, Sertoli cell count, and Leydig cell morphometry of the animal groups.

Animal Groups	Control	DM	DM+A	DM+cART	DM+A+cART
Tubule morphometry
Tubule area (µm2)	78,939 ± 692	59,561 ± 1000 ade	49,840 ± 1180 ade	65,393 ± 722 ae	75,870 ± 849
Tubule diameter (µm)	310.2 ± 1.3	269.7 ± 2.35 ade	233 ± 3.12 ade	284.7 ± 1.38 ae	302.8 ± 1.55
Epithelial height (µm)	97.52 ± 0.62	89.25 ± 1 ae	64.51 ± 1.37 abde	92.29 ± 0.8 ae	96.31 ± 0.78
Lumen diameter (µm)	97.97 ± 3.54	79.53 ± 2.84 ace	112.1 ± 3.25	81.79 ± 3.16 ace	107.4 ± 3.86
Testicular somatic cells
Sertoli cell count	23.61 ± 0.24	22.41 ± 0.41	9.64 ± 0.2 a	22.39 ± 0.34	22.39 ± 0.33
Leydig cell nuclear (LCN)
LCN Diameter (µm)	5.85 ± 0.04	4.82 ± 0.06 ade	4.24 ± 0.07 abde	5.16 ± 0.06 ae	5.52 ± 0.05 a
LCN Volume (µm3)	110 ± 2.46	67.67 ± 2.63 ade	48.43 ± 2.07 abde	79.8 ± 2.65 ae	94.09 ± 2.46 a

In the same row, different superscripts ^a, ^b, ^c, ^d, and ^e represent significant differences (p < 0.05) as analyzed by the Bonferroni’s multiple comparison test: ^a significantly different compared to the control group, ^b significantly different compared to DM, ^c significantly different compared to DM+A, ^d significantly different compared to DM+cART, and ^e significantly different compared to DM+A+cART. Key: DM, diabetes; DM+A, diabetes and alcohol; DM+cART, diabetes and combination antiretroviral therapy; DM+A+cART, diabetes and alcohol and combination antiretroviral therapy.

). Further, the DM and DM+A groups showed significant decreases (p < 0.0001) in the tubular area and diameter compared to the DM+cART group. The DM, DM+A, and DM+cART treated groups had significant reductions in the epithelial height relative to the control group (p < 0.0001, p < 0.0001, and p = 0.0010, respectively) as well as in comparison with the DM+A+cART group (p < 0.0001, p < 0.0001, and p = 0.0273, respectively). However, a nonsignificant reduction (p > 0.05) was detected in the epithelial height of the DM+A+cART group relative to the control group. The epithelial height of the DM+A group was significantly reduced (p < 0.0001) relative to the DM and DM+cART treated groups. Significant decreases were found in the lumen diameter of the DM and DM+cART treated groups relative to the control group (p = 0.0049 and p = 0.0167) and in comparison with the DM+A (p < 0.0001), and DM+A+cART groups (p < 0.0001 and p = 0.0002, respectively) (Table 2).

3.5. Germinal Epithelium Cells

The DM and DM+A+cART groups showed significant increases (p < 0.0001) in spermatogonia relative to the control group (Figure 1). However, in the DM+A and DM+cART treated groups, spermatogonia decreased significantly (p < 0.0001) relative to the control group and the rest of the treated groups. Further, the spermatogonia content was significantly decreased in the DM+A group (p < 0.0001) compared to the DM+cART group. The spermatocyte count was significantly decreased in all treated groups relative to the control group (DM, p = 0.0316; DM+A, p < 0.0001; DM+cART, p < 0.0001; and DM+A+cART, p < 0.0001). The DM+A and DM+cART treated groups showed significant decreases in spermatocytes relative to the DM (p < 0.0001 for both) and DM+A+cART (p < 0.0001 and p = 0.0068, respectively) groups. In addition, the spermatocyte count was significantly decreased in the DM+A group (p < 0.0001) relative to the DM+cART group (Figure 1). All treated groups showed significant reductions in round spermatids relative to the control group (DM, p = 0.0023; DM+A, (p < 0.0001; and DM+cART, p < 0.0001; and DM+A+cART, p < 0.0001), but the round spermatid content of the DM+A treated group reduced significantly (p < 0.0001) relative to the other treated groups. Furthermore, significant decreases in elongated spermatids were observed in all treated groups relative to the control group (p < 0.0001), and the elongated spermatid contents of the DM, DM+A, and DM+cART treated groups were significantly reduced relative to the DM+A+cART group (p = 0.0004, p < 0.0001, and p = 0.0250, respectively). Additionally, the DM+A group had a significantly decreased (p < 0.0001) elongated spermatid count relative to the DM and DM+cART treated groups (Figure 1).

3.6. Testicular Somatic Cells (Sertoli and Leydig Cells)

The number of Sertoli cells reduced significantly (p < 0.0001) in the DM+A treated group relative to the control group and all other treated groups (Table 2). Significant reductions in the Leydig cell nuclear diameter (LCND) were found in all treated groups relative to the control group (DM, p < 0.0001; DM+A, p < 0.0001; DM+cART, p < 0.0001; and DM+A+cART, p = 0.0004). The LCND of the DM, DM+A, and DM+cART treated groups was significantly reduced (p < 0.0001) relative to the DM+A+cART group. Additionally, the LCND of the DM group was significantly reduced (p = 0.0001) relative to the DM+cART group, and the LCND of the DM+A treated group was reduced significantly (p < 0.0001) relative to the DM and DM+cART groups. Furthermore, the Leydig cell nuclear volume (LCNV) was significantly reduced in all treated groups (p < 0.0001) relative to the control group. In the DM, DM+A, and DM+cART treated groups, the LCNV reduced significantly relative to the DM+A+cART group (p < 0.0001, p < 0.0001, and p = 0.0004, respectively). The LCNV of the DM group reduced significantly (p = 0.0051) relative to the DM+cART group, and the DM+A group had a significantly reduced (p < 0.0001) LCNV relative to the DM and DM+cART treated groups (Table 2).

3.7. Histopathological Evaluation

Johnsen’s testicular score reflects the progress of spermatogenesis and the number of sperm cells. All treated groups showed reductions in the Johnsen’s score, but only the DM and DM+A treated groups had significantly reduced Johnsen’s scores relative to the control group (p = 0.0268 and p = 0.0002, respectively,

Table 3. Median Johnsen’s scores and histological changes observed in animal groups.

Animal Groups	Control	DM	DM+A	DM+cART	DM+A+cART
Johnsen’s score Histological changes	8.81	8.44 a	4.04 a	8.61	8.62
Lifting of the epithelium	0.04	0.04	0.00	0.13	0.58 c
Epithelial sloughing	0.00	0.08	0.63 ad	0.00	0.29 ad
Karyolysis	0.00	0.00	0.25 abe	0.25 abe	0.00

In the same row, different superscripts ^a, ^b, ^d, and ^e represent significant differences (p < 0.05) as analyzed by Dunn’s multiple comparison test: ^a significantly different compared to the control group, ^b significantly different compared to DM, ^c significantly different compared to DM+A, ^d significantly different compared to DM+cART, and ^e significantly different compared to DM+A+cART. Key: DM, diabetes; DM+A, diabetes and alcohol; DM+cART, diabetes and combination antiretroviral therapy; DM+A+cART, diabetes and alcohol and combination antiretroviral therapy.

). It is noteworthy that the great reduction in the Johnsen’s score in the diabetic animals treated with alcohol (DM+A group) indicates disruption of the spermatogenesis progress and diminished spermatozoa production. This conforms with the severe germ cell layer loss and spermatogenesis arrest observed in this group. Further, the histology of the control group revealed a normal histoarchitecture characterized by approximately rounded tubules surrounded by a smoothly contoured basement membrane, a discretely arranged germinal epithelium with a series of spermatogenetic cells, and an interstitium consisting of blood vessels, connective tissue, Leydig cells, myoid cells, and macrophages. On the other hand, seminiferous tubule lesions of varying severity were observed in the testes of the treated animals (Figure 2). Comparisons for the lifting of epithelium, epithelial sloughing, and karyolysis were made across the animal groups (Table 3). Insignificant increases in the lifting of the epithelium were detected in the DM+cART and DM+A+cART treated groups relative to the control group, but the lifting of the epithelium in the DM+A+cART group was significantly increased (p = 0.0063) in comparison to that of the DM+A group. Epithelial sloughing was significantly increased in the DM+A and DM+A+cART treated groups relative to the control (p = 0.0100 and p = 0.0460) and DM+cART (p = 0.0100 and p = 0.0460) groups. The DM+A and DM+cART treated groups showed significantly increased karyolysis compared to the control (p = 0.0019 and p = 0.0003), DM (p = 0.0026 and p = 0.0004), and DM+A+cART (p = 0.0026 and p = 0.0005) groups. Additionally, changes, such as a widened intercellular space, extensive tubular atrophy, and ghost cells, were predominant in the DM+A group (Figure 2C,H). Germ cell pyknosis was mainly recorded in the DM and DM+A treated groups. Furthermore, an increased germ cell size and germinal epithelium derangement were observed in the DM+A and DM+A0+cART treated groups (Figure 2).

3.8. Seminiferous Tubule Basement Membrane (STBM) Thickness

The basement membrane surrounding the seminiferous tubule was identified as a thin PAS-positive stained sheet-like structure with a single layer of myoid cells (Figure 3). A significant increase in the thickness of the STBM was found in the DM+A and DM+cART treated groups relative to the control (p < 0.0001 and p = 0.0002, respectively), DM (p < 0.0001 for both), and DM+A+cART (p < 0.0001 and p = 0.0490, respectively) groups. Additionally, the STBM thickness of the DM+A treated group was significantly increased (p < 0.0001) relative to the DM+cART treated group (Figure 3).

3.9. Testicular Capsule Thickness

The testicular capsule, also referred to as the tunica albuginea, is composed of mostly collagen fibers braced with a network of myoid cells (Figure 3). The thickness of the capsule was significantly increased in the DM+A and DM+A+cART treated groups in comparison with the control group (p = 0.0002 and p = 0.0055, respectively). The DM and DM+cART treated groups showed an insignificant (p > 0.05) decrease and increase in the capsule thickness, respectively, compared to the control griyo, but the capsule thickness increased significantly (p = 0.0002) in the DM+cART group relative to the DM group (Figure 2).

3.10. Interstitial Connective Tissue

The interstitial connective tissue found between seminiferous tubules and surrounding the tubules consists of collagen, reticulin, and elastin fibers (Figure 4). All treated groups showed significant reductions (p < 0.0001) in collagen fibers relative to the control group. The DM+A treated group had significantly less collagen fibers compared to DM (p = 0.0141) and DM+A+cART (p = 0.0103) groups. The reticulin fiber content was significantly decreased in all treated groups relative to the control group (DM, p = 0.0015; DM+A, p = 0.0102; DM+cART, p = 0.0035; and DM+A+cART, p < 0.0001). Further, relative to the control group, the elastin fiber content was reduced significantly in all treated groups (DM, p = 0.0171; DM+A, p = 0.0047; DM+cART, p < 0.0001; and DM+A+cART, p = 0.0004) (Figure 4).

3.11. Reproductive Hormone Profile

The serum luteinizing hormone level decreased significantly (p = 0.0142) in the DM+A+cART group relative to the control group, whereas the luteinizing hormone level of the DM+A group was significantly increased relative to the other treated groups (DM, p = 0.0035; DM+cART, p = 0.0027; and DM+A+cART, p = 0.0002) (Figure 5). Significant increases in the serum follicle-stimulating hormone content were recorded in the DM, DM+A, and DM+A+cART treated groups relative to the control group (p = 0.0073, p = 0.0004, and p = 0.0069, respectively). Further, the follicle-stimulating hormone level of the DM+A group was significantly increased (p = 0.0176) relative to the DM+cART group. Additionally, in comparison to the control group, the serum testosterone level decreased significantly in the treated groups (DM, p = 0.0102; DM+A, p = 0.0166; and DM+A+cART, p = 0.0016). There were no significant differences in the serum inhibin B level between the control and treated groups; however, the inhibin B level of the DM+A+cART treated group was significantly increased (p = 0.0057) relative to the DM+cART treated group (Figure 5).

3.12. Androgen Receptor (AR) Immunoexpression and Staining Intensity

Testicular AR was expressed in the Sertoli cells, Leydig cells, pericytes, and myoid cells of the control and all treated animals, with exception of the DM+A treated group where the expression of AR was only detected in very few Sertoli cells (Figure 6). In the testes of all treated groups (DM, DM+A, DM+cART, and DM+A+cART), Sertoli cell androgen receptor (SAR) expression was significantly reduced relative to the control group (p = 0.0009, p < 0.0001, p < 0.0001, and p < 0.0001, respectively). However, in the DM+A treated group, SAR expression was reduced significantly (p < 0.0001) relative to the other treated groups. Additionally, the expression of SAR in the DM+A+cART group was reduced significantly (p = 0.0049) relative to the DM group. The results also showed that the SAR intensity was reduced significantly (p < 0.0001) in all treated groups relative to the control group, but the SAR intensity in the DM+A treated group was reduced significantly (p < 0.0001) compared to the other treated groups (Figure 6). The Leydig androgen receptor (LAR) expression in all treated groups was decreased significantly (p < 0.0001) relative to the control group. The expression of LAR in the DM+A treated group was significantly decreased (p < 0.0001) relative to the other treated groups, but the LAR expression in the DM+A+cART treated group was decreased significantly relative to the DM (p < 0.0001) and DM+cART (p = 0.0346) groups. Equally, all treated groups showed significant reductions in the LAR intensity relative to the control group (DM, p = 0.0001; DM+A, p < 0.0001; DM+cART, p = 0.0054; and DM+A+cART, p = 0.0158). Further, the LAR intensity in the DM+A treated group was significantly reduced (p < 0.0001) relative to the other treated groups (Figure 6). Furthermore, all the results reported in this study are summarized in

Table 4. Summary of the study’s results.

		TREATED GROUPS
PARAMETERS		DM	DM+A	DM+cART	DM+A+cART
Fasting glucose		↑ *	↑ *	↑ *	↑ *
Testis morphometry
Testis weight		↓	↓ *	↓	↓
Testis volume		↓	↓ *	↓	↓
Testis size		↓	↓ *	↓	↓
Testicular component area fractions (AF)
Epithelial AF		↓ *	↓ *	↓ *	↓
Lumen AF		↓ *	↑	↓ *	↓
Interstitial space AF		↑ *	↑ *	↑ *	↑ *
Connective tissue AF		↑ *	↑ *	↑ *	↑ *
Tubule morphometry
Tubule area		↓ *	↓ *	↓ *	↓
Tubule diameter		↓ *	↓ *	↓ *	↓
Epithelial height		↓ *	↓ *	↓ *	↓
Lumen diameter		↓ *	↑	↓ *	↑
Spermatogenic cell count
Spermatogonia		↑ *	↓ *	↓ *	↑ *
Spermatocytes		↓ *	↓ *	↓ *	↓ *
Round spermatids		↓ *	↓ *	↓ *	↓ *
Elongated spermatids		↓ *	↓ *	↓ *	↓ *
Testicular somatic cells
Sertoli cell count		↓	↓ *	↓	↓
Leydig cell nuclear	-Diameter	↓ *	↓ *	↓ *	↓*
	-Volume	↓ *	↓ *	↓ *	↓*
Histopathological evaluation
Johnsen’s score		↓ *	↓ *	↓	↓
Lifting of epithelium		VSC	↓	↑	↑
Epithelial sloughing		↑	↑ *	VSC	↑ *
Karyolysis		VSC	↑ *	↑ *	VSC
Connective tissue
Seminiferous tubule basement membrane thickness		↓	↑ *	↑ *	↑
Testicular capsule thickness		↓	↑ *	↑	↑ *
Collagen		↓ *	↓ *	↓ *	↓ *
Reticulin		↓ *	↓ *	↓ *	↓ *
Elastin		↓ *	↓ *	↓ *	↓ *
Reproductive hormones
Luteinizing hormone		↓	↑	↓	↓ *
Follicle stimulating hormone		↑ *	↑ *	↑	↑ *
Testosterone		↓ *	↓ *	↓	↓ *
Inhibin B		↑	↑	↓	↑
Androgen receptor (AR)
Sertoli cell AR		↓ *	↓ *	↓ *	↓ *
Leydig cell AR		↓ *	↓ *	↓ *	↓ *

* Significantly different compared to the control group. ↑ = increase; ↓ = decrease. Key: VSC, value same as the control group; DM, diabetes; DM+A, diabetes and alcohol; DM+cART, diabetes and combination antiretroviral therapy; DM+A+cART, diabetes and alcohol and combination antiretroviral therapy.

.

4. Discussion

The increases in the prevalence of diabetes [8], alcohol abuse [9], and the use of the combination antiretroviral therapy (cART) regimen due to an increase in HIV/AIDS disease [2] in males of reproductive age in a millennium burdened with a global decline in male fertility are of great concern [1]. Moreover, previous studies have shown the negative impacts of diabetes [14,36], alcohol abuse [15,17], and cART use [18,20] on the male reproductive system. In addition to the rising incidences of these conditions and their specific devastating adverse effects on male reproductive capacity and sexual efficiency, they are intricately connected and can occur simultaneously in an individual [10,11,12,13]. Such a conundrum might elicit severe testicular dysfunction. The present report highlights the impact of diabetes, alcohol, and cART interactions on the male reproductive capacity and male fertility.

Morphometry and histopathology are widely used tools to determine perturbations in the testis structure and function [19,37] and were utilized in the present study. Although the mean morphometric and histopathological parameters of the treated groups were statistically different from those of the control group, we observed that certain seminiferous tubules were severely altered, some were slightly altered, but others were unaltered, suggesting variable tubular effects of the treatment. This implies that the degree of disruption to spermatogenesis may differ in various tubules; thus, normal spermatogenesis is sustained in some tubules regardless of the treatment toxicity, as previously demonstrated by Azu et al., 2014 [19]. Our findings reveal alterations in the evaluated morphometry parameters of the testis, with significant decreases in mean testicular and tubule parameters, component area fractions, and germinal epithelium cells in treated animals relative to the control group. These results indicate severe testicular architecture perturbations and the derangement of cellular coherency due to toxicity associated with the existence of alcohol, cART, or both in diabetic individuals. However, while the significant increases in testicular interstitium area fraction may complement the decreases in the tubule diameter, the increased lumen diameter in the alcohol- and cART-treated groups may reflect the highly significant decreases recorded in the epithelial height and the overall adverse effects on germinal epithelial cells. Further, the significant reductions in the tubular area and diameter point to tubular shrinkage in the treated groups and suggest the occurrence of tubular degenerative changes following diabetes treatments [38,39] as well as evidence of disturbed spermatogenesis. Additionally, the significant increment in the connective tissue area fraction recorded across the treated groups may imply a buildup of fibrosis in the interstitium, which corresponds to earlier reports [40] in infertile men with impaired testicular integrity and cellular (Leydig, macrophage, myoid) function. Conversely, the morphometric findings conformed with the histopathological observations and the seminiferous tubule lesions across the treatment groups, suggesting there is consistency in the treatment effects on the testicular histological profile. We previously reported seminiferous tubule atrophy, epithelial derangement, germ cell depletion, and distorted interstitium in the testes of rats treated with alcohol, cART, or both (alcohol+cART) [34]. Further, Nna et al., 2019 [36] demonstrated similar observations in diabetic rat testes. Histological and morphometric testicular alterations are morphological indicators of disruption of the testicular structure integrity and its attendant dysregulation impact on spermatogenesis [41,42]. Overall, alcohol use by diabetics was associated with the greatest toxicosis in all morphometry parameters.

The function of the testicular somatic cells (Sertoli and Leydig cells) is essential for the development of the testis and the regulation of spermatogenesis [43]. Sertoli cells nurse germ cells and regulate the seminiferous tubule milieu [44]. Our results revealed a nonsignificant reduction in the Sertoli cell number in the testes of diabetic animals and in all treated groups, but the marked significant reduction in the diabetic + alcohol treated group (DM+A) with extensive germ cell depletion suggests a disruption of the critical role of Sertoli cells in the process of spermatogenesis. The nonsignificant reduction of the Sertoli cell number in diabetic, diabetic plus cART, and diabetic plus alcohol plus cART groups suggests minimal perturbations in the Sertoli cell function, which may be linked to the high contents of the antiapoptotic makers Bcl-w and Bcl-xL [45,46]. However, disruptions of the Sertoli cell structure impair cell function, which affects the blood–testis barrier and can lead to germinal epithelial detachment into the lumen [47], as recorded in the present study, especially in the diabetic plus alcohol group. 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, Giannessi et al. (2015) [55] observed necrotic Leydig cells in the testes of mice treated with alcohol, and Olasile et al. (2018) [27] reported reduced LCNVs in rats treated with cART. Further, Kianifard 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 seminiferous 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 rete testis [33]. Therefore, the observed significant increase in the thickness of the seminiferous 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, Olojede 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, reticulin, and elastin) were reduced in treated groups relative to the control group, which is consistent with previous reports that documented reductions in collagen and reticulin 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. We recorded significant decreases in the luteinizing hormone (LH) (except for the DM+A group) and testosterone (TT) levels in all treated groups, particularly in the DM+A+cART group. There were significantly increased levels of follicle-stimulating hormone (FSH) and insignificant increases in inhibin B (INHB) levels in all treated groups, except for the DM+cART group, which showed a nonsignificant increase in FSH and a decrease in the INHB level. These perturbations of the hormonal profile reflect disturbances of endocrine homeostasis [43]. In addition, high levels of FSH indicate an impairment of testis function, particularly in relation to the Sertoli cells that secrete INHB, which regulates FSH production [68]. Many similarities and discrepancies exist in hormonal profile results, with increased levels of LH and FSH [69] and LH and TT [70] shown in diabetic individuals; decreased levels of TT shown in diabetics [71], alcohol consumers [16], and cART users [72]; and decreased FSH levels shown in diabetics [51]. Remarkably, Rossi et al., 2020 [73] found that Sertoli cells exposed to bisphenol A had increased INHB production. While such observed hormonal imbalances may reflect differences in the research design, methods, and duration of treatment exposure, they all express adverse functional perturbations of the HPG axis. Furthermore, the balance among the HPG axis hormones is regulated via a negative feedback mechanism, whereby the TT and INHB hormones produced by testicular cells (Leydig and Sertoli cells respectively) modulate the pituitary secretion of LH and FSH, respectively [67,74]. Conversely, decreased levels of both LH and TT and an increase in the FSH level when the level of INHB is not reduced, as observed in this study, might imply that the treatments have disrupted the HPG axis feedback mechanism. Additionally, the recorded hormonal imbalances conform with the observed Sertoli and Leydig cell perturbations and consequently lead to spermatogenesis and steroidogenesis impairments.

Furthermore, human studies have reported altered reproductive hormone profiles in HIV-infected males under antiretroviral therapy [75,76], which is associated with the high prevalence of gynecomastia and sexual dysfunction in this category of patients [77]. In addition, factors associated with a poor sperm quality, including reduced ejaculate volume and sperm motility, spermatozoa dysmorphology, and increased sperm nuclear fragmentation were recorded following long-term treatment with cART [78,79]. Conversely, although the impact of changes in the levels of reproductive hormones has been studied extensively in relation to male reproductive dysfunction, investigations on the changes in their receptors are scant, despite their vital roles in successful male reproduction and fertility [44,67]. The androgen receptor is expressed by testicular somatic cells, such as Leydig, Sertoli, myoid, and pericyte cells, and it regulates the structures and functions of these cells and mediates the actions of TT on spermatogenesis [80,81]. The observed significant reductions in the number and staining intensity of Sertoli and Leydig cells expressing the androgen receptor in all treated groups and, interestingly, the more severe losses recorded in the diabetes-alcohol treated group (DM+A), are suggestive of testicular toxicity and consequent dysfunction. Our findings are consistent with previous reports on decreased androgen receptor expression in diabetic rat testes, resulting in weak androgen receptor signaling and leading to testicular and sexual dysfunction [82,83]. Other studies on organochlorinated pesticides, industrial chemicals, and plasticizers have reported similar testicular toxic effects [28,84], including several seminiferous tubule impairments in rats treated with the diacerein drug [85]. Moreover, hypocellular testes with the arrest of spermatogenesis were reported in androgen-receptor-knockout animal models [81,86]. In addition, transgenic androgen receptor disruption studies have revealed the involvement of androgen receptor signaling in the maintenance of spermatogonia proliferation, the integrity of the blood–testis barrier, spermatogenesis progression, and spermiation [87]. This indicates the crucial roles of the androgen receptor in spermatogenesis failure, subfertility, and infertility [88,89]. Therefore, the downregulation of androgen receptors in treated animals could have led to alterations in the testicular morphometry, structural integrity, and spermatogenesis, as observed in the current study.

It is interesting that the testicular histoarchitecture was more severely affected in the diabetic + alcohol treated group (DM+A group) than in the other treated groups. This adverse effect may not be unrelated to the uncontrollable high blood glucose levels associated with alcohol abuse in diabetics, which could have led to the exacerbated testicular damage observed [90,91]. However, overall, diabetes + alcohol and/or cART or both variably adversely affected the parameters evaluated. The lesser adverse effects observed in the DM+A+cART treated group may be due to the shared metabolism pathway (cytochrome P450) for alcohol and cART, leading to a diminished impact when alcohol and cART are used concurrently by diabetics [12,92] or due to the altered bioavailability of cART (tenofovir) in diabetics, as was previously reported [93].

5. Conclusions

This study revealed that the use of alcohol or/and cART by diabetics causes testicular toxicosis conditions ranging from morphometric and structural derangements to cellular and seminiferous tubule lesions, including the loss of germ and Sertoli cells, reduction of the volume of the Leydig cell nucleus, an adversely reduced androgen receptor concentration, and dysregulated reproductive hormones following treatment. This points to derangements in spermatogenesis and the male reproductive capacity/fertility. The greatest toxicity was documented in diabetic animals treated with alcohol (DM+A group) and the lowest toxicity occurred in diabetic animals treated with cART (DM+cART group). The results further suggest that the interaction arising from the co-presence of cART and alcohol in diabetic individuals could mildly diminish their independent effects due to their common cytochrome P450 metabolic pathway. Notably, the study findings are based on the use of Atripla and should not be generalized to other cART regimens. Based on the observations of this study, the authors recommend that further human/clinical studies are carried out to document the impact of taking alcohol and cART concurrently on the male reproductive system in diabetic individuals.