Original Article |
Corresponding author: Mina Pencheva ( mina.pencheva@mu-plovdiv.bg ) © 2022 Mina Pencheva, Yvetta Koeva, Ekaterina Pavlova, Nina Atanassova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Pencheva M, Koeva Y, Pavlova E, Atanassova N (2022) Stage specific expression of angiotensin-converting enzyme and thickened lamina propria in relation to male fertility. Folia Medica 64(1): 41-48. https://doi.org/10.3897/folmed.64.e59819
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Introduction: The testis is an immune privileged organ that provides a specific environment for germ cell development. Various factors responsible for inflammatory changes can lead to deterioration of the immune tolerant model found in the testis. As a result, the thickness of the proper membrane of seminiferous tubules changes and the process of spermatogenesis is disturbed.
Aim: The purpose of the present study was to find the connection between the changes in the level of testis-specific isoform of angiotensin-converting enzyme (tACE) expression and the morphological changes of the seminiferous tubule wall of the testis in patients with infertility.
Materials and methods: The study included 24 infertile men who underwent a testicular biopsy. Routine histological techniques, immunohistochemical reactions for tACE, α-smooth muscle actin, and morphometric analysis were performed to examine the biopsy preparations.
Results: By using testicular biopsy to diagnose patients with infertility, a stage-specific pattern of the processes associated with thickened proper membrane of seminiferous tubules was established and a decreased or absent spermatogenic activity was observed.
Conclusions: A significant increase in the proper membrane thickness of the seminiferous tubules in the testis was found in patients with infertility. This finding shows that the processes take place gradually over time, correlating with the degree of pathology, and that changes do not depend on the factors causing them. We also found that the degree of proper membrane thickening correlated with disturbances of spermatogenesis, using tACE expression as a marker for spermatogenic epithelium differentiation.
angiotensin-converting enzyme, infertility, membrana propria, myofibroblasts, spermatogenesis
Most of the testicular parenchyma is made up of the seminiferous tubules where the process of spermatogenesis occurs. The seminiferous tubules develop during puberty with the formation of the tubular lumen. The seminiferous tubule consists of the spermatogenic epithelium and the proper membrane, membrana propria (MP). The MP of the seminiferous tubules, also known as the peritubular lamina, surrounds the cellular elements. Differentiated epitheliocyti sustentans (Sertoli cells, ES) and incessantly proliferating spermatogenic cells are placed inside the tubular lumen.[
Human MP is composed of several layers of myoid cell and extracellular matrix between them.[
The myoid cells provide structural integrity of the tubules and participate in the regulation of spermatogenesis.[
The steroid producing cells, endocrinocyti interstitiales (Leydig cells, EI) prominent in the inter-tubular space, produce and secrete one of the essential male reproductive hormones, the testosterone.[
The testicular angiotensin-converting enzyme (tACE) is a shorter isoform of ACE, which is specifically expressed only in the testes. It has been found that this enzyme plays an essential role in spermatogenesis and the stages of spermatid differentiation in the testes.[
Immunochemical analysis of adult rat testis also revealed a stage-specific pattern of tACE expression in the cytoplasm of postmeiotic germ cells. tACE could serve as a marker for germ cell depletion in experimental and pathological conditions.[
The purpose of the present study was to find a connection between the changes in the level of tACE expression and the morphological alterations in the seminiferous tubule wall in patients with infertility.
Testicular biopsies from patients with history of infertility and azoospermia were included in this study by testicular sperm extraction (TESE) and using a standard open surgical biopsy technique which is performed at the same time as ART. The testicular tissue obtained was provided by Malinov Specialized Surgical Hospital in Sofia. The study was conducted between 2018 and 2019, and included 24 infertile men (age range 21-42 years) with azoospermia (obstructive azoospermia [n=12], non-obstructive azoospermia [n=3], varicocele [n=6], and cryptorchidism [n=3]). All study patients gave their written informed consent to undergo a testicular biopsy. Tissue samples were removed until spermatozoa were identified or 3–4 biopsy pieces were extracted from each testis. The samples were fixated in Bouin’s solution after being washed and placed in 70% alcohol, embedded in paraffin blocks, cut into slices using an automatic paraffin microtome (Leica 2055), dewaxed, and rehydrated in descending series of ethanol (100%, 95%, 70%) and distilled water.
The morphological analysis of the biopsy material based on the MP morphology and spermatogenic activity allowed each biopsy preparation to be assigned to one of four groups showing increasing pathology. The four groups the biopsy material was allocated to according to Volkmann et al.[
The testicular biopsies were fixed at room temperature in Bouin’s solution and embedded in paraffin. Five-μm-thick paraffin sections were stained with haematoxylin and eosin (H&E) and Heidenhain’s azan[
The sections were stained with Mayer’s hematoxylin (5 min) and differentiated in tap water (20 min). After completion of the differentiation, the sections were stained with water-soluble eosin for 5 minutes. The subsequent stained sections were dehydrated again in ascending grades of ethanol (70%, 95%, and 100%), cleared in xylene (2×10 min) and covered with Canadian balm.
For Azan staining, the slides were incubated in aniline alcohol (0.1 ml aniline dissolved in 100 ml 90% ethanol) for 5 min and in prewarmed azocarmine G (0.1% azocarmine G, 1% glacial acetic acid) for 10 min at 60°C. The specimens were chilled for 10 min, washed with H2O, differentiated with aniline alcohol (for 5 to 10 minutes), briefly washed with acetic alcohol and H2O, and incubated in 5% phosphotungstic acid for 2 hours. The next steps were incubation in aniline blue/orange G solution (0.25% aniline blue, 1% orange G, 4% glacial acetic acid) for 2 hours and then differentiation using 96% alcohol.
Sections were deparaffinised, then subjected to antigenic detection of the epitopes with citrate buffer, and an endogenous peroxidase blockade was made with hydrogen peroxidase (3%), a kit (ref: No. BBK 120, Scy Tek, USA) was used to block the endogenous biotin аnd a reagent to block non-specific binding (Superblock, Scy Tek), followed by incubation for 24 hours at 4°C with anti-goat АСЕ - 1:300 (sc-12187, Santa Cruz Biotechnology Inc. USA) and monoclonal anti-α smooth muscle actin (A-2547, Sigma) 1:5000 - NRS/TBS/BSA, next incubated with secondary antibody: biotinylated anti-goat (No. AGL015 Scy Tek., USA) for 10 min. The reaction was visualized with 3,3΄-diaminobenzidine tetrachloride (DAB, ScyTek Lab. Inc., USA); counterstaining was performed with Mayer’s hematoxylin. As negative controls, sections in which the primary antibodies were replaced by a buffer solution (PBS) were used. Microphotographs were performed with Nikon Microphot SA microscope (Japan), combined with Camedia-5050Z digital camera (Olympus, Japan) at ×100 and ×400 magnification.
The measurements were performed using Olympus DP - Soft 4.1 software, Japan. Morphometric measurement was performed on paraffin sections measuring the wall thickness of an average of 50 seminiferous tubules per patient. An average of six measurements of the seminiferous tubule wall thickness were carried out at ×200 magnification on Azan staining slices and on a positive immunohistochemical reaction for actin (α-SMA). The entire wall thickness - BM + MP was measured, as well as its components. The groups were determined based on the seminiferous tubule thickness, according to the classification of Volkmann et al.[
The data were analysed using SPSS 19.0. Statistical significance between experimental groups was determined by the independent samples t-test and Mann-Whitney U test, the differences were considered significant at p<0.05. Correlative analysis was performed. Data are presented as mean± SEM.
All methods used in the study were approved by the Scientific Ethics Committee at the Medical University of Plovdiv with decision No. Р-1166/15.04.2016.
Following the H&E and Heidenhain’s azan staining of paraffin sections, the histological analysis of group 1 revealed clearly distinguishable structures - seminiferous tubules containing approximately three layers of myofibroblast cells in MP, ES, and normal spermatogenesis with all types of germ cells - spermatogonia, primary spermatocytes, round and elongated spermatids, mature sperm. The immunohistochemical analysis of group 1 revealed expression of the tACE protein only in the spermatogenic epithelium of the seminiferous tubules. Heidenhain’s azan staining was performed to visualize myofibroblast cells (red nuclei and light pink cytoplasm) forming MP. We found a change in the type of MP - as thickness was increased, the relief was changed, and the irregular outline contour was increased. In the testicular interstitium, clusters of EI and blood vessels were well distinguished.
In the comparative analysis of the preparations in groups 2, 3, and 4, the MP thickening was pronounced, accompanied by deposition of extracellular matrix between the myofibroblast layers, which was well visualized by H&E and azan staining, as well as with immunoreactivity for α-SMA.
In group 2, we found MP thickening accompanied by deposition of extracellular matrix between the myofibroblast layers, which was visualized by H&E and azan staining and immunoreactivity for α-SMA. Decreased tACE expression in individual seminiferous tubules was reported. Disorganization and exfoliation of the spermatogenic epithelium and its location in the lumen were observed in some of the tubules. In the testicular interstitium, disintegration of the characteristic arrangement of EI was observed, accompanied by lymphocyte infiltration.
In group 3, the two layers of myofibroblasts could be distinguished, an inner row of myofibroblasts and several outer layers of myofibroblasts with ECM deposited between them, with variable thickness in the individual tubules. Using α-SMA, the thickness of the seminiferous tubule wall was measured, and we found significant thickening in individual tubules probably due to the larger amount of ECM between the myofibroblast layers. Single EI and lymphocyte infiltration were observed in the interstitium.
In group 4, tACE was not visualized in seminiferous tubules with strongly thickened MP, in which only ES were present in groups 3 and 4. Immunoreactivity for α-SMA was visualized only in the outer layer, composed of several layers of myofibroblasts which were in contact with the cells in the interstitium, a dense layer of ECM of variable thickness was located inside it, and in certain tubules the lumen was strongly narrowed due to the deposition of ECM. The spermatogenic epithelium was in direct contact with the ECM in the seminiferous tubules. Only blood vessels were visualized in the testicular interstitium (Fig.
Seminiferous tubules (groups 1-4) – immunoreactivity for tACE and α-SMA, haematoxylin and eosin (H&E), and azan (A) staining; ×100, ×400. Seminiferous tubules of group 1 - (tACE), (H&E), and (A) show normal MP thickness and preserved spermatogenesis; (→) immunoreactivity for α-SMA visualizes myofibroblast cells in MP. By group 2 - reduced tACE expression in individual seminiferous tubules accompanied by disorganization and exfoliation of the spermatogenic epithelium in the lumen (→). (A) and α-SMA show an increase in MP thickness. Lymphocytic infiltration in the testicular interstitium is found. Group 3 - low tACE expression in single round spermatids, (H&E) and (A) outline distinct ES. α-SMA in MP clearly distinguishes two layers of myofibroblasts - outer and inner (→); ECM (*) is deposited between them. There is no specific organization of EI, presence of lymphocyte infiltration. Group 4 - tACE is not visualized; (H&E) and (A) show a reduced lumen with single ES; α-SMA visualizes the absence of an inner layer of myofibroblasts, and only the presence of an outer layer, composed of several myofibroblast layers.
Fig.
Statistical analysis of morphometric data on immunoreactivity for α-SMA is presented in Fig.
Analysis of the MP thickness of the seminiferous tubule wall. A. Azan staining; B. α-SMA immunoreaction; ** p<0.05 when compared to group 1.
Fig.
The statistical analysis showed a very strong negative correlation between MP thickness and the percentage of tACE-positive cells in the seminiferous tubules in both types of measurements - tACE-azan (r=−0.714, p=0.01) and α-SMA-tACE (r=−0.529, p=0.01). The higher value in the correlation between tACE-azan is probably due to the methodology and the non-specific deposition of azan on collagen fibrils in adjacent structures (Figs
Analysis of the percentage of tACE immunopositive cells in the seminiferous tubule wall; ** p<0.05 when compared to group 1.
One of the most common causes of male infertility is a past medical history of inflammatory processes in the testis and the epididymis. Inflammation of the testis leads to changes in the seminiferous tubule wall and impairment of spermatogenesis. In case of pathological changes, MP cells increase the secretion of various molecules of the extracellular matrix. This results in impaired communication between the myofibroblasts and the spermatogenic epithelium, which is essential for maintaining spermatogenesis. This association has been demonstrated in in-vitro studies of ES and peritubular cells of rats, which indicates that MP cells are closely related to cell populations in the walls of the seminiferous tubules and the interstitial cells.[
Inflammation leads to lymphocyte infiltration and reactive T-cells activation in the testicular interstitium adjacent to the myofibroblasts,[
The results of the present study support the above-mentioned data and are indicative for the crucial role of the inflammation processes of the testis and the epididymis in the etiology and pathogenesis of male infertility. It is important to note that in our classification of biopsy materials, the stages of the pathological changes in the seminiferous tubules can be traced, regardless of the etiology. In the present study, group 1 included biopsies in which variations of intact tubules with preserved spermatogenic activity were observed. The histopathological changes we found in the second group preparations were characterized by massive lymphocyte infiltration in both the testicular interstitium and the seminiferous tubules, these observations corresponded to the results reported by previous studies.[
In the present study, the immunoreactive expression for tACE was used to report the stage-specific sperm differentiation because it is well known that tACE is exclusively expressed in differentiating haploid spermatids, with a significant role in the stages of spermiogenesis.[
The data analysis showed an increase in the MP thickness corresponding to the degree of pathology; these changes developed gradually over time following a certain pattern which did not depend on the causes. It was also found that the degree of MP thickening correlated with the disturbance in spermatogenesis, using tACE expression as a marker for spermatogenic epithelial differentiation.
The research is funded by Medical University of Plovdiv, project No. 08/2015.