EFFECTS OF SILDENAFIL ON THE TESTES OF THE NONDIABETIC AND DIABETIC ADULT ALBINO RATS (Light and Electron Microscopic Study)

INTRODUCTION

Sildenafil citrate (SC) was introduced for treatment of angina pectoris, but it was observed that it induces marked penile erection. In males with mild to severe erectile dysfunction (ED) without an organic cause, the medication has been proven to be beneficial ¹.

SC inhibits the breakdown of cyclic guanosine monophosphate (cGMP) in the corpus cavernosum of the penis through cGMP-specific phosphodiesterase type 5 (PDE5). This causes erection by promoting smooth muscle relaxation, vasodilation, and increased blood flow into the spongy tissue of the penis².

SC induces degenerative changes in the seminiferous tubules as well as interstitial histological alterations, which finally might lead to an arrest of the spermatogenesis³.

In human, the testis is the primary sex organ of males and is formed mainly of stroma and parenchyma. It is a tubular gland that performs both endocrine and exocrine activities⁴.

Diabetes mellitus (DM) is a critical disease. End-organ damage will occur in many systems, including the genitourinary system, if DM not well controlled⁵.

Alloxan is an organic compound has the molecular formulae, C4H2N2O4. Alloxan is a diabetogenic drug that is frequently used in diabetes research to examine the antidiabetic potential of both pure chemicals and plant extracts. Alloxan-induced diabetes is a kind of insulin-dependent diabetes that develops after animals are exposed to alloxan⁶.

This work aimed to reveal the effect of sildenafil citrate in high and toxic doses on the histological structure of the normal and diabetic rat testes.

MATERIALS AND METHODS

Fifty adult male albino rats aging 12-14 weeks and weighing 150-200 grams were used. The study was done at Animal House, Faculty of Pharmacy, Al-Azhar University, Cairo, between July and September 2021.

Experimental Design:

The rats were randomly divided into three groups and had free access to water and food:

Group I (control group): 10 rats received distilled water daily for 2 months.

Group II (Nondiabetic group): 20 rats was divided into 2 equal subgroups: II-A and II-B.

Group II-A: received intraperitoneal injection (IPI) of SC 9 mg/kg/d for 2 months⁷.

Group II-B: received IPI of SC 13.5 mg/kg/d for 2 months⁸.

Group III (diabetic group): 20 rats were divided into 2 equal subgroups: III-A and III-B. Both subgroups had free access to water and food and were treated with a single dose of IPI of 150 mg/kg of alloxan to induce diabetes. Seventy-two hours later, blood glucose level was determined to confirm induction of diabetes (normal blood glucose level of the rats is 50-95 mg /dl fasting and 70-130 post prandial). Rats with blood glucose level >250 mg/dl were considered diabetic and included in the experiment Then,

Group III-A: received IPI of SC 9 mg/kg/d for 2 month⁷.

Group III-B: received IPI of SC 13.5 mg/kg/d for 2 months⁸.

Chemicals:

The chemicals used in this work were:

- SC 100mg tablets were purchased from Pfizer Egypt Pharmaceutical Company and the tablets were crushed and diluted in 50 ml of sodium chloride, so each 1 ml of sodium chloride were contained 2mg of sildenafil citrate.

- Alloxan was present in the form of alloxan hydrate in aqueous solution 1.2 g in 12 ml of normal saline. It was purchased and manufactured by Sigma Aldrich Company, UK.

Methods:

1) Testes specimens collection:

Rats in all groups were sacrificed after 2 months. The collected testes of each group were divided into two subgroups for processing.

The first subgroup was processed for paraffin sectioning and stained by Hematoxylin and eosin stain (H&E) and Masson’s trichrome stain. The other half of testes were processed for transmission electron microscopic

2) Light and electron microscopic processing:

The collected testes were divided transversely, and 2 mm thick section of each specimen was immersed in 2.5% glutaraldehyde solution. Then, they fixed for 2 hours in 1% osmium tetraoxide at 4°C. Then after washing the tissue three times in distilled water, the specimens were prepared for dehydration, infiltration, ultramicrotomy. The tissues then were stained by uranyl acetate and lead citrate for transmission electron microscopic processing (TEM).

Then, the rest of each dissected testis was preserved in 10% buffered formalin and then processed for paraffin sections then stained by H&E and also Masson's trichrome stain for light microscopic examination ^9,10.

RESULTS

1) External appearance and mortality:

Examination of the external appearance of the testes revealed that there was no difference between the testes excised from all groups. Regarding mortality, no deaths were recorded among the animals of all groups.

2) Effect of SC on testes histology:

A- Light microscopic finding:

Control (Gp1): testis showed normal tunica albuginea with normal sub-capsular blood vessels, normal-sized tubules with normal tunica propria, normal germinal lining and complete spermatogenic layers, and normal interstitium with Leydig cells (Figure. 1-A).

Gp II (non-diabetic + SC): testis showed thick tunica albuginea with mildly congested sub-capsular blood vessels, scattered normal-sized and distorted tubules with quietly normal tunica propria, normal germinal lining and complete spermatogenic layers, and normal interstitium with Leydig cells. These changes were more evident in high-dose (Figure. 1-C) than in low-dose sildenafil (Figure. 1-B).

Gp III (diabetic + SC): testis showed markedly thickened tunica albuginea with congested sub-capsular blood vessels, scattered small-sized distorted tubules with irregular tunica propria, thin germinal lining and diminished spermatogenic layers, and interstitium with Leydig cells. These changes were more evident in high-dose (Figure. 1-E) than in low-dose sildenafil (Figure. 1-D).

B- Electron microscopic Finding:

Control )Gp1( : testis showed seminiferous tubules with spermatogonia resting on normal tunica propria with normal myoid cells, nucleus with dispersed chromatin and rounded mitochondria, primary spermatocytes with nucleus showing dispersed chromatin, well-formed endoplasmic reticulum small cytoplasmic vacuoles, Sertoli cells with indented nucleus showing prominent nucleoli and numerous lysosomes (Figure. 2-A).

Gp II (non-diabetic + SC): testis showed seminiferous tubules with spermatogonia resting on thick irregular tunica propria with smaller myoid cells, nucleus with dispersed chromatin in the periphery and swollen and distorted mitochondria with irregular cristae, primary spermatocytes with small nucleus, large cytoplasmic vacuoles, swollen endoplasmic reticulum and distorted mitochondria with irregular cristae, and rounded and elongated spermatids, and portions of distorted spermatozoa with no acrosomal cap, directed towards Sertoli cell with nucleus showing prominent nucleolus. These changes were more evident in high-dose (Fig. 2-C) than in low-dose sildenafil (Figure. 2-B).

Gp III (diabetic + SC): testis showed seminiferous tubules with scattered primary spermatocytes showing small nucleus showing clumped chromatin in the periphery, small, rounded mitochondria and large cytoplasmic vacuoles, many rounded and elongated spermatids with less-formed acrosomal cap, and portions of well-formed spermatozoa with well-formed acrosomal cap directed towards Sertoli cells with numerous rounded mitochondria and numerous lysosomes. These changes were more evident in high-dose (Figure. 2-E) than in low-dose sildenafil (Figure. 2-D).

Fig 1: A photomicrograph of a section of the testis of A) control group showing normal sized tubules (T) with normal germinal lining (blue arrow), full layers of germinal lining (black arrows) and normal interstitium (red arrow) occupying spaces between the tubules. B) Non-diabetic + SC 9mg group showing quietly normal-sized tubules and normal interstitium (red arrow). Some of tubules appear normal with normal germinal lining and complete spermatogenic layers (black arrows), while others have distorted and separated germinal lining (blue arrows). C) Non-diabetic + SC 13.5mg group showing thick tunica albuginea (black arrow) with mildly congested sub-capsular blood vessels (yellow arrow), quietly normal-sized tubules with distorted & spaced germinal lining (red arrow), and normal interstitium (blue arrow). D) Diabetic + SC 9mg group showing disturbed tunica albuginea (blue arrow), sub-capsular accumulation of fluid (red arrow) and widely separated distorted tubules (black arrows). E) Diabetic + SC 13.5mg group showing widely spaced distorted seminiferous tubules with obliterated lumen (black arrows) (H & E, X 200).

Fig 2: An electron photomicrograph of sections of the testis of A) control group showing seminiferous tubules with spermatogonia (Sg) resting on normal tunica propria (yellow arrow) with nucleus showing dispersed chromatin (red arrow) and rounded mitochondria (blue arrow), primary spermatocytes (Psp) with swollen mitochondria (M) and small cytoplasmic vacuoles (white arrow), Sertoli cells (SC) with indented nucleus showing prominent nuclei (orange arrow). B) Non-diabetic + SC 9mg group showing seminiferous tubules with primary spermatocytes (Psp) with nucleus (N) showing dispersed chromatin (yellow arrow), rounded mitochondria (red arrow) and large cytoplasmic vacuoles (white arrow), and few distorted spermatozoa with less formed acrosomal cap (blue arrow) directed towards Sertoli cell (SC) with numerous lysosomes (orange arrow). C) Non-diabetic + SC 13.5mg group showing seminiferous tubules with spermatogonia (Sp) resting on thick tunica propria (yellow arrow) with myoid cell (blue arrow) and nucleus with dispersed chromatin (red arrow), primary spermatocytes (Psp) with nucleus (N) showing dispersed chromatin (white arrow), and Sertoli cell (SC) and small rounded mitochondria (orange arrow). D) Diabetic + SC 9mg group showing seminiferous tubules with spermatogonia (Sp) resting on thick irregular tunica propria (yellow arrow) with smaller myoid cell (blue arrow), nucleus (N) with clumped chromatin in the periphery (red arrow), primary spermatocytes (Psp) with small nucleus (N) and clumped chromatin (white arrow), large cytoplasmic vacuoles (orange arrow), and distorted mitochondria with irregular cristae (M). E) Diabetic + SC 13.5mg group showing seminiferous tubules with spermatogonia (Sg) resting on markedly thickened irregular tunica propria (yellow arrow) and markedly swollen mitochondria with irregular cristae (red arrow), primary spermatocytes (Psp) with small nucleus (N) and clumped chromatin in the periphery (blue arrow), and Sertoli cell with nucleus showing prominent nucleolus (white arrow). (TEM, X 6000).