Bioscience Journal | 2023 | vol. 39, e39033 | ISSN 1981-3163 1 Alluanan Adelson do Nascimento SILVA1 , Jessica Santana DE OLIVEIRA1 , Fernanda Carolina Ribeiro DIAS1,2 , Rosana Nogueira DE MORAES3 , Elizabeth Neves DE MELO4 , Pierre Castro SOARES1 , Valdemiro Amaro DA SILVA JUNIOR1 1 Department of Veterinary Medicine, Universidade Federal Rural de Pernambuco, Recife, Pernambuco, Brazil. 2 Department of Structural Biology, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil. 3 Department of Physiology, Universidade Federal do Paraná, Curitiba, Paraná, Brazil. 4 Department of Anatomy, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil. Corresponding author: Alluanan Adelson do Nascimento Silva alluanan@gmail.com How to cite: SILVA, A.A.N., et al. The effectiveness of insulin therapy in the functional and morphological recovery of leydig cells in rats submitted to diabetes mellitus. Bioscience Journal. 2023, 39, e39033. https://doi.org/10.14393/BJ-v39n0a2023-65096 Abstract The effects of systemic insulin administration at different concentrations on the testicular tissue of diabetic adult rats, induced by streptozotocin, are evaluated by the morphological analysis of spermatogenic process. Twenty-four adult male rats were divided into 1) Control Group: they received citrate buffer, by intraperitoneal injection; 2) Diabetic Group: induced by intraperitoneal injection of streptozotocin (60 mg. kg-1 of body weight); 3) Insulin 50%: induced diabetes treated with half of standard dosage of insulin; 4) Insulin 100%: induced diabetes treated with standard dose of insulin. After eight weeks, animals were weighted and anesthetized; testicles were removed and processed in resin. Body and testicular weight of diabetic rats decreased when compared to that of control. Parameters increased with insulin therapy. Testosterone levels were low in diabetic animals but rates recovered after insulin therapy. Nuclear diameter and volume of Leydig cells decreased in diabetic rats although they significantly increased after insulin therapy. Results showed that the administration of insulin in diabetic rats promoted a protective effect of testicular parenchyma, enhancing efficient recovery on testosterone levels and increase in daily sperm production. Keywords: Diabetes Mellitus. Insulin Therapy. Leydig Cells. Seminiferous Tubule. Testis. 1. Introduction Diabetes mellitus is a metabolic disease characterized by the persistence of high serum glucose levels from unfavorable changes in the production and/or action of insulin in the body (Maresch et al. 2018; Rodacki et al. 2022). It is a critical health problem that has been intensifying worldwide, with many adverse effects over time, such as retinopathy, nephropathy, cardiovascular symptoms, and sexual dysfunction (Joshi et al. 2017; Hajam and Rai 2019). Diabetic men experience reproductive damage, but the mechanisms that cause this damage are still unclear (Ricci et al. 2009; Joshi et al. 2017; He et al. 2021). Diabetes affects the testicular parenchyma by causing a reduction in testicular weight, delay in the seminiferous epithelium cycle, depletion of germ cells, and a reduction in the height of the seminiferous epithelium in animals induced to experimental diabetes (Mallick et al. 2007; Kianifard 2011; Guo et al. 2016). THE EFFECTIVENESS OF INSULIN THERAPY IN THE FUNCTIONAL AND MORPHOLOGICAL RECOVERY OF LEYDIG CELLS IN RATS SUBMITTED TO DIABETES MELLITUS https://orcid.org/0000-0001-6452-5234 https://orcid.org/0000-0003-1641-1384 https://orcid.org/0000-0002-6530-4684 https://orcid.org/0000-0002-0816-4371 https://orcid.org/0000-0002-0630-9485 https://orcid.org/0000-0002-5680-3940 https://orcid.org/0000-0002-1894-7337 Bioscience Journal | 2023 | vol. 39, e39033 | https://doi.org/10.14393/BJ-v39n0a2023-65096 2 The effectiveness of insulin therapy in the functional and morphological recovery of leydig cells in rats submitted to diabetes mellitus These changes in the testicular tissue relate to the dysregulation of the hypothalamus-pituitary- gonadal (HPG) axis, usually reported in diabetes, thus reducing the serum levels of essential hormones for good spermatogenic performance, such as the follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (Ballester et al. 2004). FSH regulates Sertoli cells, affects sperm production, and is present at low levels in diabetic individuals (Condorelli et al. 2018). However, there is a decline in LH levels, which regulate the function of Leydig cells and, consequently, promote adverse effects on testosterone production (Ballester et al. 2004). Testosterone is mostly produced by Leydig cells from the use of cholesterol molecules in a process called steroidogenesis. Normal testosterone levels are crucial for normal spermatogenesis and are directly related to secondary sexual characteristics (Goto et al. 2020). Thus, reductions in testosterone levels are usual in diabetic individuals and may relate to Leydig cell function damage (Ballester et al. 2004; Ricci et al. 2009). Studies indicate a relationship between insulin and Leydig cells, requiring normal levels of this hormone for efficient testosterone production (Ballester et al. 2004). Therefore, the exogenous insulin protocol is extensively used in diabetic individuals, representing the main form of disease control by reducing blood glucose levels (Rodacki et al. 2022). However, despite studies reporting that insulin therapy improves tubular and sperm parameters (Hajam and Rai 2019; Samadian et al. 2019), studies evaluating Leydig cells are still scarce. Considering that the integrity and functionality of these cells are essential for the normal development of spermatogenesis, this study aimed to assess the effect of administering different insulin concentrations on the morphology of Leydig cells in diabetic individuals. 2. Material and Methods Experimental Design Twenty-four adult male Wistar rats (Rattus norvegicus, var. albinus), 70 days old and weighing 200- 300 g, were randomly divided into four groups (n=06): Control Group (CG); Diabetic Group (DG); 50% Insulin (Ins50); 100% Insulin (Ins100). Each group had free access to pelleted food and water until the end of the experiment. They were maintained in a 12-hour reverse light/dark cycle, with controlled humidity (50%) and temperature (22°C), in the vivarium of the Anatomy Department of the Federal University of Pernambuco (UFPE), Brazil. The present study was approved by the Ethics Committee for Animal Experimentation of UFPE (38/12 on 06/11/2013). After the rats had fasted for 14 hours, diabetes was induced with a single intraperitoneal injection of streptozotocin (60 mg. Kg-1). The Control Group received citrate buffer (vehicle) in the same way. The animals were considered diabetic when they manifested glucose levels above 200 mg.dl-1. The glycemic index was verified weekly and before euthanasia by collecting blood from the caudal vein (Glucometer Kit). The diabetic rats were treated with a daily subcutaneous administration (5 UI) of NPH (Neutral Protamine Hagedorn) human insulin. After the onset of diabetes, insulin was administered daily for eight weeks (Gobbo et al. 2015). Next, the animals were weighed and anesthetized intraperitoneally with ketamine (70 mg/kg) and xylazine (10 mg/kg), followed by deepening of anesthesia with sodium thiopental (80 mg/kg) (Pereira et al. 2018). The animals were subjected to perfusion procedures with a cannula inserted in the left heart ventricle. First, the vessel was cleared with 0.9% NaCl saline solution for two minutes. The animals were then perfused with 4% glutaraldehyde in a phosphate buffer solution for 40 minutes. All testicles were collected, weighed, and post-fixed with the same fixative. The samples were embedded in resin, and 4-µm- thick fragments were stained with hematoxylin-phloxine. A quantitative testicular analysis was performed, and the gonadosomatic index (GSI = [testicular weight/body weight] x 100]) was calculated (Oliveira et al. 2022). Bioscience Journal | 2023 | vol. 39, e39033 | https://doi.org/10.14393/BJ-v39n0a2023-65096 3 SILVA, A.A.N., et al. Testicular morphometry Volumetric density of testicular compartments Volume densities of the testicular parenchyma were obtained with a 441-intersection grid placed under a light microscope. Fifteen randomly chosen sites (6615 points) were scored for each animal at a 400x magnification. The intersection points over the testicular parenchyma tissue were considered as follows: tubular compartment (tunica propria, seminiferous epithelium, and lumen) and intertubular space (Leydig cells, connective tissue, and blood and lymphatic vessels). The volume of each testicular component (ml) was established by Oliveira et al. (2022) (Figure 1). Figure 1. A - Photomicrographs showing the tubular compartment at stage VII. A – control group; B – diabetic group with the vacuoles on seminiferous tubules (black arrow) and the cell death of germination cells (white arrow); C – the diabetic group treated with 50% insulin; D – the diabetic group treated with 100% insulin (400x). Bars: 13.72 µm. Tubular diameter, height of seminiferous epithelium and total length of seminiferous tubules Tubular diameter and height of the seminiferous epithelium were measured at a 100x magnification in an image-capture system connected to the light microscope. Fifteen randomly chosen round tubules were measured at various stages of the seminiferous cycle. The rates are the means between two diametrically opposite measurements. The total length of seminiferous tubules per testis (in meters) was estimated with the ratio between the absolute volume of seminiferous tubules in the testis by R² (R = tubular diameter/2) and the π value (Amann and Almquist 1962). Bioscience Journal | 2023 | vol. 39, e39033 | https://doi.org/10.14393/BJ-v39n0a2023-65096 4 The effectiveness of insulin therapy in the functional and morphological recovery of leydig cells in rats submitted to diabetes mellitus Score of spermatogenic cells, population of Sertoli cells and daily spermatic production Spermatogonia, preleptotene/leptotene spermatocyte I, pachytene spermatocyte I, round spermatids, and Sertoli cells nucleoli were counted in five round seminiferous tubules at stage VII in each animal. Then, the number obtained was adjusted (Amann and Almquist 1962). The mean Sertoli nucleolar cell diameter (5 per animal) was measured at a 1000x magnification, and the population was calculated with the product of the adjusted number of Sertoli cells and the total length of seminiferous tubules in micrometers, divided by slice thickness. Daily sperm production per testis was obtained (Oliveira et al. 2022). Nuclear diameter, cell volume and population of Leydig cells Thirty nuclear profiles were captured at a 1000x magnification and analyzed. The nuclear diameter was obtained with two diametrically opposite measurements (Oliveira et al. 2022). The volumetric density (%) of Leydig cells was measured with a 441-point reticule (locked to the microscope) at a 1000x magnification. One thousand points were counted, including the nucleus and cell cytoplasm. The volume of each Leydig cell was determined with mathematical models by nuclear diameter rates and proportion in the testis (Oliveira et al. 2022). The Leydig cell population was obtained with volume rates of each cell, volumetric density (%), and total volume (ml) occupied by Leydig cells in the testis. Plasma testosterone Testosterone was quantified with enzyme immunoassay (Munro et al. 1991) using a polyclonal anti- testosterone antibody (R 156⁄7 1:7500 dilution) obtained from Coralie Munro University of California, Davis (Davis, CA, USA). Statistical analysis The results were blinded to normality assumptions with the Kolmogorov-Smirnov test and subjected to base-10 logarithmic transformation and ANOVA, with the GLM procedure in the SAS software (Statistical Analysis Systems Institute Inc). Means were compared with the minimum significant difference (m.s.d.) of the Student-Newman-Keuls (SNK) test at a 5% significance level. Testosterone was subjected to logarithmic transformation, and Pearson´s correlation analyses (r) were performed to verify relationships among variable pairs (Preece and Hills 1982). High-intensity correlation occurred when r >0.60, medium intensity when 0.30