Abstract
Background
As an important glucocorticosteroid hydrocortisone has been widely used in clinical practice. Even though a series of side effects induced by long-term administration of hydrocortisone are known, but side effects related to testicular injury of juvenile mice are still unknown. Here we explored the damage of the testicular tissue of male juvenile mice induced by long-term administration of hydrocortisone.
Methods
To damage the testicle, male ICR juvenile mice were orally given hydrocortisone (69 µmol/kg/day, once a day for 16 consecutive days) for establishing mouse testicular injury model. To get normal testicle, male ICR mice were orally given CMC-Na (10 mL/kg/day, once a day, for 16 consecutive days). The morphological change was shown by the testicular interstitium and seminiferous tubules, as well as the number of the spermatogenic cells. To get insight into the potential mechanism of damaging testicular tissue the level of IL-2, IL-6 and TNF-α of the corresponding samples were tested by ELISA experiment.
Results
Long-term use of hydrocortisone resulted in a number of changes, such as the damage of testicular tissue, the decreasing of the number of spermatogonia in all three phases, a selective effect on spermatocytes in proliferative phase only, the decreasing of the number of sperm cells in all three phases, the down regulation of IL-2, IL-6 and TNF-ɑ both in the serum and testicular tissue of male juvenile mice.
Conclusions
Hydrocortisone is widely used to treat a series of diseases clinically, and some side reactions are well known. To the best of our knowledge, testicular injury for male juvenile mice was a newly founded side reaction caused by long-term administration of hydrocortisone. Moreover, the decrease of serum level of IL-2, IL-6 and TNF-ɑ could be the molecular mechanism, and may be used as potential biomarker to monitor the progression of testicular injury for male adolescent patients of receiving long-term hydrocortisone. Avoiding testicular damage is crucial for male adolescent patients receiving hydrocortisone, and the finding is of importance for improving the clinical applications of hydrocortisone.
Keywords: Hydrocortisone, Mouse testis, Injury, Morphology, Spermatogenic cells, Cytokines
Introduction
Hydrocortisone is an important regulatory hormone and is essential for maintaining normal physiology. Hydrocortisone is involved in a series of physiological processes, such as participating metabolism [1], participating water and electrolyte balance [2], participating immune response [3], participating growth [4], relating to cardiovascular function [5], affecting mood and cognitive function [6], affecting reproduction and development [7–9]. Clinically, hydrocortisone is widely used to prevent hemodynamic deterioration caused by protamine sulfate during surgery in adult cardiac surgery patients [10], to improve oxygenation during respiratory deterioration in premature infants without obvious adverse reactions [11, 12], to treat hypotension in critically ill neonates [13], to treat patients with primary adrenal insufficiency caused by autoimmune diseases [14]. Hydrocortisone is also widely used for replacement therapy in particular [15–17]. However, the long-term administration [18] and the cumulative dosage [19] lead hydrocortisone to face many challenges resulted from adverse reactions, such as diabetes and arterial hypertension [20, 21], adrenal atrophy [22], infection risk [23], and osteoporosis [14, 24, 25].
Recently, the tissue injury is correlated with the inflammatory factor [26–31]. In this context, the aberrant expression of interleukin 6 (IL-6) is considered as the important inflammatory mechanism underlying adverse effects of capecitabine (CAP) in hand-foot syndrome (HFS); while interleukin 2 (IL-2) and tumor necrosis factor-α (TNF-α) are known as the multifunctional master pro-inflammatory anticancer cytokines [32–35]; CD4 and CD8 cells are known as important immune cells of human immune system and to be closely related to interleukin [36].
In our investigation, we observed that long-term administration of hydrocortisone damaged the testis of male juvenile mice. This paper focused on the testis damage of male juvenile mice induced by long-term administration of hydrocortisone, thereby focused on the change of IL-2, IL-6 and TNF-α in vivo.
Materials and methods
Chemicals and reagents
Hydrocortisone (HC, Sigma), 1×PBS (0.01 M, pH7.4, KeyGen Biotech), 4% paraformaldehyde (Solarbio), all reagents and solvents (Sinopharm Chemical Reagent Co., Ltd.) for this work were obtained commercially and used without further purification, unless otherwise specified.
Animals
Male ICR mice (12 ± 2 g, 21-day old) were purchased from the Laboratory Animal Center of Capital Medical University. All evaluations were conducted in accordance with the protocol. The protocol was reviewed and approved by Ethics Committee of Capital Medical University (Ethical Approval Number: AEEI-2021-202). The committee assured that the animal welfare was maintained in accordance with the requirements of Animal Welfare Act and NIH Guide for Care and Use of Laboratory Animals. Male ICR mice (12 ± 2 g, 21-day old) were divided into HC damaged group (10 mice) and normal testicular group of SHAM mice (10 mice), and for the morphological study both testicles were sampled.
Our pre-experiment showed that 16-day treatment of HC was suitable for damaging the testicular tissue of juvenile mice. Thus to establish HC damaged testicular mice, male ICR mice (12 ± 2 g, 21-day old) were orally given HC (69 µmol/kg/day, once a day, for 16 consecutive days). This dose of HC was resulted from both of our pre-experiment and the maximum therapeutic dose for humans been 10 mg/kg/day. To get normal testicular, Sham mice were orally given CMC-Na (sodium carboxymethyl cellulose, 10 mL/kg/day, once a day, for 16 consecutive days). On day 17, the orbital blood of the mice was collected for ELISA experiments. To sacrifice the mice were deeply anesthetized with isoflurane and then euthanized. Both testis samples were then collected, the right and the left testicles were fixed with 4% tissue fixation solution (4% paraformaldehyde, Solarbio, Beijing, China) for 24 h, dehydrated, paraffin embedded, sectioned, and hematoxylin-eosin (HE) stained for preparing pathological section; the right and the left testicles were processed routinely to prepare homogenates for ELISA experiments.
ELISA experiment
Into the orbital blood the solution of sodium citrate in normal saline (3.8%) was added on a ration of one to nine (v/v), and then the blood samples were centrifuged (3,000 g, 15 min, 4 °C). Our pre-experiment showed that 16-day treatment of HC did not change the levels of testosterone and cortisol in the serum and the testicular tissue of juvenile mice of juvenile mice. In this case our ELISA experiments were focused on IL-2, IL-6, IL-8, TNF-ɑ, CD4 and CD8. The supernatant was obtained, the ELISA experiments were performed by using the manufacturer’s protocols, and the interleukin-2 (IL-2, F2698-A, Fankew, Shanghai, China), interleukin-6 (IL-6, F2163-A, Fankew, Shanghai, China), interleukin-8 (IL-8, F2123-A, Fankew, Shanghai, China), and the tumor necrosis factor (TNF-α, F21632-A, Fankew, Shanghai, China), as well as the immune cells CD4 (F2656-A, Fankew, Shanghai, China) and CD8 (F2657-A, Fankew, Shanghai, China) were measured, accordingly.
Into the testis ultrapure water was added to homogenize, and then the homogenate samples were centrifuged (3,000 g, 15 min, 4 °C). The supernatant was then obtained, the ELISA experiments were performed by using the manufacturer’s protocol to measure the level of IL-2, IL-6, IL-8 and TNF-α, as well as CD4 and CD8 in the testis.
ELISA experiments were performed by using ELISA kit (mouse IL-2, IL-6, IL-8, TNF-α, CD4 and CD8 ELISA kit, China) and the 96-well plate coated with the enzyme. The well containing 980 µL of the serum of the samples or the supernatant of homogenate samples were incubated at 37 °C for 3 min. The plate was incubated at 37 °C for 120 min, and the solvent was removed. To each well 100 µL of biotin labeling antibody (from the kit) were added, incubated at 37 °C for 60 min, the solvent was removed, and washed for three times. To each well 100 µL of horseradish peroxidase labeling avidin were added, incubated at 37 °C for 60 min, the solvent was removed and the plate washed five times with PBS. To each well 90 µL substrate was added, incubated at 37 °C in dark for 20 min-coloration. To each well 50 µL of stop solution was added to stop the coloration. Within 15 min the OD values of the wells were tested at 450 nm and the concentration of mouse IL-2, IL-6, IL-8, TNF-α, CD4 and CD8 was calculated.
Histological morphology
Our pre-experiment showed that 16-day treatment of HC did not change sperm concentration, motility, and percentage of live and dead sperm of juvenile mice. In this case the analyses of sperm concentration, motility, as well as the percentage of live and dead sperm were not involved. The effect of HC on the histological morphology of mouse testis was carried out by comparing the pathological section of HC damaged testes with those of normal testes. Thus HC damaged testis and normal testis were excised, the pathological Sects. (5–8 μm in thickness) were prepared and subsequently were fixed in formaldehyde. In the preparation of eosin dye solution, 0.5 g of hematoxylin powder and 24 g of ammonium alum were dissolved in 70 mL of distilled water to prepare solution I, and 31 g of NaIO, 5 mL of distilled water, 30 mL of glycerin and 2 mL of concentrated acetic acid were mixed to prepare solution II. The solutions I and II were evenly mixed and filtered to provide the filtrate as water soluble eosin dye, then 0.5 g of water soluble eosin dye solution was dissolved in 100 mL of distilled water to obtain eosin dye solution.
When making pathological section of mouse testis, mouse testis was put into the pre-prepared 10% formaldehyde fixing solution to fix the testis, so as to maintain the original shape of the testis. When dehydrating formaldehyde-immobilized testis, mouse testis was placed in an embedded box and rinsed with running water for 30 min to remove the formaldehyde-immobilized fluid from the testis, and then dehydrated in ethanol solutions of varying concentrations, from low to high. Finally, the mouse testis dehydrated with ethanol solution was treated with xylene in the embedding box, so that the ethanol in the mouse testis was replaced by xylene for wax embedding. Mouse testis replaced with ethanol by xylene was first immersed in wax in a molten paraffin box, and then buried. The paraffin immersed in the mouse testis cooled, solidified into clumps, and the mouse testis were hardened and were cut into slices 5 to 8 microns thick on a microtomer. To avoid creasing the slices, the slices were ironed in hot water, then they were placed onto glass slides and dried in a 45 °C incubator.
Slices of mouse testis on slides dried in a 45 °C incubator were soaked in xylene for de-paraffin, then successively soaked in ethanol (from high to low concentration) and distilled water for staining. Mouse testicular sections were stained in hematoxylin staining solution for 3–8 min, washed with tap water, differentiated in ethanol containing 1% hydrochloric acid for no more than 10 s, rinsed with tap water, returned to blue with 0.6% ammonia, and rinsed with running water for 1 h. Slice was dyed in eosin solution for 1–3 min and rinsed under running water for 1 h. Eosin-stained slices were treated in ethanol (70%, 80%, 90% and 100%) for 10 s, in xylene for 1 min, and in the fume hood for additional 5 min waiting for sealing. When sealing, by using a straw into the slice 1 drop of neutral gum was added try to cover the slice completely. Finally, the section was sealed with a glass sheet, and the glue can be affixed with a label after drying for later research.
Morphology of the cells of the last stage of sperm formation
According to the literature [37, 38], for mice twelve stages were involved in the process of sperm development and maturation. According to the morphology of the cells of the last stage of sperm formation in the seminiferous epithelium, the stages of spermatogenesis in the seminiferous epithelium can be roughly divided into three phases, i.e. the proliferative phase, the meiotic phase and the terminal phase of differentiation.
In the proliferative phase the cells of the last stage of sperm formation were immature, having small number and long oval shape. The cells of the last stage of sperm formation were mainly located in the villi extending from the basement membrane to the official cavity. There were more spermatocytes, which obviously showed a fusiform division process. The nuclei of spermatogonium were significantly larger and darker than those of other spermatogenic cells.
In the meiotic phase, the spermatid mature spermatozoa transformed from immature long oval shape to mature thin pointed long sperm. In this transforming process, the number of spermatid mature spermatozoa became larger, and the sperm of both immature and mature stages were included, and the spermatid mature spermatozoa was closer to the inside of the official cavity. At this time, the spermatocyte color was darker, and each spindle monomer can be clearly seen. The color of spermatogonia was deeper than that of proliferative stage, and the nuclei were not larger than that of proliferative spermatogenic phase.
In terminal phase of differentiation, the cells of the last stage of sperm formation mainly showed mature spermatozoa, mostly as fine grained strips, converging in the middle of the lumen. At this stage mature spermatozoa completely entered the central lumen, and were transported to the next. The color of spermatocyte was lightened. The color of spermatogonia was deeper, the nucleus was more obvious, and the surface spermatogenesis was transitioned to proliferative spermatogenesis.
The proliferative phase, the meiotic phase and the terminal phase of differentiation were known as the coherent spermatogenic processes. The process was distinguished according to the morphology and location of spermatogenic cells. When sperm entered the lumen, the terminal phase of differentiation will enter the proliferative phase again and carried out a new cycle spermatogenic process. The spermatogenesis process of each convoluted tubule was independent. The statistical analysis of the morphology was carried out accordingly. The number of spermatogenic cells was quantified based on the histological micrographs and by using Image J software.
Jhonsen score based histopathological damage
To make the interpretation of HC-damage towards the testicular tissue of the mice more robust Johnsen score was applied. For this object thirty tubules from each mouse were scored at 480× magnification. Each tubule received a score from 1 to 10 based on in the testicular seminiferous tubules whether spermatozoa, spermatids, spermatocytes, spermatogonia, germ cells and Sertoli cells were presence or absence. Better spermatogenesis was associated with higher Johnsen,s score, while lower score was associated with significant dysfunction.
Statistical analysis
Statistical analyses were performed by using SPSS V. 22.0 (IBM, Chicago, USA) and GraphPad Prism 8 (La Jolla, CA, USA). All experiment data were described as the mean ± SD. The statistical significance between two groups was determined by using Student’s t-test, while one-way analysis of variance (ANOVA) and Dunnett’s test were used to evaluate the significance among multiple groups. The two-sided P value of less than 0.05 was considered statistically significant.
Results
Long-term use of HC damaging testicular tissue of juvenile mice
In order to investigate the specific damage induced by long-term administration of HC, we prepared pathological sections of the testicles of normal mice and HC treated juvenile mice. The pathological sections showed that HC can change the testicular morphology of the mice.
The pathological sections were shown with Fig. 1. For the pathological sections of the testicles of normal mice, in the testicular interstitium there were round interstitial cells with eosinophilic cytoplasm, while for the pathological sections of the testicles of mice damaged by HC, in the testicular interstitium there were almost no round interstitial cells with eosinophilic cytoplasm. Besides, the interstitial spaces of the damaged testicular tissue were reduced and the interstitial cells were irregular in shape.
Fig. 1.
The testicular tissue sections: Normal a) the normal testis section under a 20× microscope; HC injured a) HC injured testis section under a 20× microscope; Normal b) the normal testis section under a 100× microscope; HC injured b) HC injured testis section under a 100× microscope; Normal c) the normal testis section under a 480× microscope; HC injured c) HC injured testis section under a 480× microscope. d) The Johnsen score of seminiferous tubular cross-sections in normal and HC injured male mice.The cells with black arrow represent the interstitial cells and the cells with red arrow represents the lumen
The seminiferous tubules were shown with the seminiferous epithelium. Figure 1 (normal a-c) show that on the surface of the seminiferous epithelium of the normal seminiferous tubules, there are mature elongated spermatids distributed in pairs and elongated spermatids close to maturity distributed alone, as well as under the lumen there are round spermatids of the developmental stage. Figure 1 (HC injured a-c) show that on the surface of the seminiferous epithelium of HC damaged seminiferous tubules, there are no mature elongated spermatids distributed in pairs and no elongated spermatids close to maturity distributed alone, as well as under the lumen there are no round spermatids of the developmental stage. Comparing with normal seminiferous tubules, the basement membrane boundary of HC damaged seminiferous tubules becomes blurred, the villi and branches in the lumen become fewer and more disorderly, and the number of sperm is significantly decreased. Figure 1d shows that comparing with the Johnsen score of the normal seminiferous tubules the Johnsen score of HC damaged seminiferous tubules is significantly decreased. It should be indicated that in Fig. 1a-c the black arrow points the interstitial cells and the red arrow points the lumen.
Long-term use of HC and the number of spermatogonia of male juvenile mice
According to the different morphologies of the spermatogenic cells in the seminiferous epithelium, the process of producing mature sperm in the seminiferous tubules was divided into three phases, i.e. the proliferative phase, the meiotic phase and the terminal phase of differentiation.
Based on the morphology of spermatogonia and pathological sections of seminiferous tubules, the number of the spermatogonia was calculated. HC can destroy the tissue morphology of the testis and reflect the microscopic level of juvenile mice. As seen in Table 1; Fig. 2B, HC significantly decreases the number of the spermatogonia in the proliferative phase and the meiotic phase (compared with normal mice, P < 0.01), as well as significantly decreases the number of the spermatogonia in the terminal phase of differentiation (compared with normal mice, P < 0.05). This phenomenon was further visualized with Fig. 2A.
Table 1.
Effects of different spermatogenic phases on spermatogonia count
| Spermatogonia source | Spermatogonia count during spermatogenesis, Mean ± SD | ||
|---|---|---|---|
| Proliferative phase | Meiotic phase | Terminal phase | |
| Normal mice | 44.0 ± 7.3 | 26.0 ± 6.1 | 53.0 ± 8.8 |
| HC injured mice | 25.0 ± 5.7a | 18.0 ± 4.3a | 40.0 ± 15.3b |
Values were represented with the mean and standard deviation of independent experiments as shown in Fig. 2B; Statistical significance was determined with one-way ANOVA followed by Dunnett’s test; a compared with normal mice P < 0.01; b compared with normal mice P < 0.05; n = 10
Fig. 2.
Photomicrographs of the spermatogonia at three spermatogenic phases of HC treated mice versus normal mice. A The normal spermatogenic morphology (including local magnificated spermatogenic morphology) and HC injured spermatogenic morphology (including local magnificated spermatogenic morphology) at three spermatogenic phases under a 480× microscope. B The number of spermatogonia cells in the proliferative phase, meiotic phase, terminal phase of differentiation, values represent mean ± SD of independent experiments, statistical significance was determined with one-way ANOVA followed by Dunnett’s test, n = 10. The red arrow shows the location of the spermatogonia
Long-term use of HC and spermatocytes in proliferative phase of male juvenile mice
As the key cells to germ cells, the changes of the number of spermatocytes were particularly characterized. The data of Table 2; Fig. 3B show that HC selectively decreases the number of spermatocytes in the proliferative phase (compared with normal mice, P < 0.01), but not the number of spermatocytes in the meiotic phase and the terminal phase of differentiation (compared with normal mice, P > 0.05). This selectivity was further emphasized with Fig. 3A.
Table 2.
Effect of different spermatogenic phases on spermatocytes counts
| Spermatocytes source | Spermatogenic phase spermatocytes count, Mean ± SD | ||
|---|---|---|---|
| Proliferative phase | Meiotic phase | Terminal phase | |
| Normal mice | 50.0 ± 9.8 | 33.0 ± 8.4 | 34.0 ± 14.9 |
| HC injured mice | 36.0 ± 10.5a | 29.0 ± 4.5b | 39.0 ± 15.7b |
Values were represented with the mean and standard deviation of independent experiments as shown in Fig. 3B; statistical significance was determined with one-way ANOVA followed by Dunnett’s test; a compared with normal mice P < 0.01; b compared with normal mice P > 0.05; n = 10
Fig. 3.
Photomicrographs of the spermatocytes at three spermatogenic phases of HC treated mice versus normal mice. A The normal spermatocyte morphology (including local magnificated spermatocyte morphology) and HC injured spermatocyte morphology (including local magnificated spermatocyte morphology) at three spermatogenic phases under a 480× microscope. B The number of spermatocytes in three spermatogenic phases, values represent mean ± SD of independent experiments, statistical significance was determined with one-way ANOVA followed by Dunnett’s test, n = 10.The red arrow shows the location of the spermatocytes
Long-term use of HC and spermatozoa cells in three stages of male juvenile mice
The changes in the number of spermatozoa in germ cells were the key to judging testicular damage. HC damaged the testicular tissue of mice, and this kind of damage was manifested by destroying the tissue morphology of the testicles and necessarily decreased spermatozoa number in the proliferative phase, the meiotic phase and the terminal phase of differentiation. The data of Table 3; Fig. 4B show that HC significantly decreases spermatozoa number in all three spermatogenic phases (compared with normal mice, P < 0.01). Figure 4A was used to show this kind of damage more intuitively and more clearly.
Table 3.
Effect of different spermatogenic phases on spermatozoa counts
| Sperm source | Spermatogenic phase spermatozoa count, Mean ± SD | ||
|---|---|---|---|
| Proliferative phase | Meiotic phase | Terminal phase | |
| Normal mice | 115.0 ± 37.1 | 133.0 ± 29.1 | 67.0 ± 19.0 |
| HC injured mice | 75.0 ± 13.2a | 89.0 ± 20.9a | 41.0 ± 12.7a |
Values were represented with the mean and standard deviation of independent experiments as shown in Fig. 4B; statistical significance was determined with one-way ANOVA followed by Dunnett’s test; a compared with normal mice P < 0.01; n = 10
Fig. 4.
Photomicrographs of the spermatozoa at three spermatogenesis phases of HC treated mice versus normal mice. A The normal spermatozoa morphology (including local magnificated spermatozoa morphology) and HC-injured spermatozoa morphology (including local magnificated spermatozoa morphology) at three spermatogenesis phases under a 480× microscope. B Cell number of the last stage of sperm formation of all three spermatogenesis phases, values represent mean ± SD of independent experiments, statistical significance was determined with one-way ANOVA followed by Dunnett’s test, n = 10.The red arrow shows the location of the spermatozoa
Down-regulation of IL-2, IL-6, IL-8 and TNF-ɑ of male juvenile mice
The damage of HC towards the testicular tissue of mice may be reflected by serum concentration of IL-2, IL-6, IL-8 and TNF-ɑ. This consideration promoted measuring serum concentration of IL-2, IL-6, IL-8 and TNF-ɑ. The damage of HC towards the testicular tissue of mice may be reflected by testicular concentration of IL-2, IL-6, IL-8 and TNF-ɑ. This consideration promoted measuring testicular concentration of IL-2, IL-6, IL-8 and TNF-ɑ.
Table 4 shows that HC decreases serum concentration of IL-2, IL-8 (compared with normal mice, P < 0.05), IL-6 and TNF-α (compared with normal mice, P < 0.01). Table 5 shows that HC decreases testicular concentration of IL-2 and TNF-α (compared with normal mice, P < 0.05) as well as IL-6 (compared with normal mice, P < 0.01), but not IL-8 (compared with normal mice, P > 0.05). The data of Tables 4 and 5 demonstrated that HC simultaneously decreased both serum concentration and testicular concentration of IL-2, IL-6 and TNF-α of male juvenile mice. On the other hand, however, Table 5 demonstrated that HC decreased testicular concentration of IL-2, IL-6 and TNF-α of male juvenile mice only. Besides, it was worthy to mention that the interstitial damage was described in the present paper. In this case the damage of Leydig cells perhaps should be considered, which been well known to be involved in the decrease of testicular concentration of IL-2 and TNF-α. Therefore, long-term use of HC whether or not damage Leydig cells should be investigated in the further.
Table 4.
Effect of HC injury on serum cytokine concentrations in mice
| Serum source | Effect on the concentrations of the following cytokines, Mean ± SD pg/mL | |||
|---|---|---|---|---|
| IL-2 | IL-6 | IL-8 | TNF-α | |
| Normal mice | 240.8 ± 3.1 | 116.9 ± 9.9 | 96.9 ± 6.4 | 459.7 ± 22.5 |
| HC injured mice | 222.8 ± 13.9a | 98.2 ± 5.8b | 90.0 ± 1.3a | 422.3 ± 11.3b |
Values were represented with the mean and standard deviation of independent experiments; statistical significance was determined with one-way ANOVA followed by Dunnett’s test; a compared with normal mice P < 0.05; b compared with normal mice P < 0.01; n = 10
Table 5.
Effect of HC injury on testicular cytokines in mice
| Testis | Effect on the concentrations of the following cytokines, Mean ± SD pg/g | |||
|---|---|---|---|---|
| IL-2 | IL-6 | IL-8 | TNF-ɑ | |
| Normal mice | 2455.6 ± 139.4 | 1057.0 ± 73.3 | 927.9 ± 30.0 | 2455.6 ± 139.4 |
| HC injured mice | 2264.0 ± 72.4a | 878.5 ± 78.4b | 936.0 ± 20.2c | 2264.0 ± 72.4a |
Values were represented with the mean and standard deviation of independent experiments; statistical significance was determined with one-way ANOVA followed by Dunnett’s test; a compared with normal mice P < 0.05; b compared with normal mice P < 0.01; c compared with normal mice P > 0.05; n = 10
Long-term use of HC having no effect on CD4 and CD8 of male juvenile mice
The damage of HC towards the testicular tissue of mice may be reflected by serum concentration of CD4 and CD8. This consideration promoted measuring serum concentration of CD4 and CD8. The damage of HC towards the testicular tissue of mice may be reflected by testicular concentration of CD4 and CD8. This consideration promoted measuring testicular concentration of CD4 and CD8.
Table 6 shows that the damage of HC increases serum concentration of CD4 (compared with normal mice, P < 0.05), but not CD8 (compared with normal, P > 0.05). Table 7 shows that the damage of HC does not affect testicular concentration of CD4 and CD8 (compared with normal mice, P > 0.05). The data of Tables 6 and 7 demonstrated that long-term use of HC had no effect on the serum concentration of CD8 as well as the testicular concentration of CD4 and CD8 of male juvenile mice.
Table 6.
Effects of HC injury on serum CD4 and CD8 in mice
| Serum source | Serum concentration of CD4 and CD8, Mean ± SD ng/mL | |
|---|---|---|
| Serum CD4 | Serum CD8 | |
| Normal mice | 4.10 ± 0.12 | 6.41 ± 0.27 |
| HC injured mice | 4.31 ± 0.12a | 6.15 ± 0.16b |
Values were represented with the mean and standard deviation of independent experiments; statistical significance was determined with one-way ANOVA followed by Dunnett’s test; a compared with normal mice P < 0.05; b compared with normal mice P > 0.05; n = 10
Table 7.
Effect of HC injury on testis CD4 and CD8 in mice
| Testis source | Testis concentration of CD4 and CD8, Mean ± SD ng/mL | |
|---|---|---|
| CD4 | CD8 | |
| Normal mice | 40.78 ± 2.37 | 62.58 ± 3.75 |
| HC injured mice | 39.64 ± 2.08a | 59.91 ± 1.46a |
Values were represented with the mean and standard deviation of independent experiments; statistical significance was determined with one-way ANOVA followed by Dunnett’s test; a compared with normal mice P > 0.05; n = 10
Long-term use of HC having no effect on body weight and testicular weight of male juvenile mice
In order to know long-term administration of HC whether or not affect body weight and testicular weight, the body weight and the testicular weight of mice orally administered by HC for 16 days were compared with those of normal mice. Tables 8 and 9; Fig. 5 consistently show that HC does not affect the body weight and the testicular weight of the mice (P > 0.05).
Table 8.
Effect of 16-day administration of HC on body weight
| Group | Dosing concentration | Weight, Mean ± SD g |
|---|---|---|
| Normal mice | - | 32.37 ± 2.60 |
| HC injured mice | 69 µmol/kg/day | 31.01 ± 2.19a |
Values were represented with the mean and standard deviation of independent experiments as shown in Fig. 5A; statistical significance was determined with one-way ANOVA followed by Dunnett’s test; a compared with normal mice P > 0.05; n = 10
Table 9.
Effect of 16-day administration of HC on testicular weight
| Group | Left testicle weight, mean ± SD mg | Right testicle weight, mean ± SD mg |
|---|---|---|
| Normal mice | 95 ± 25 | 96 ± 26 |
| HC injured mice | 100 ± 6.9a | 101 ± 7.8a |
Values were represented with the mean and standard deviation of independent experiments as shown in Fig. 5B; statistical significance was determined with one-way ANOVA followed by Dunnett’s test; a compared with normal mice P > 0.05; n = 10
Fig. 5.
Effect of HC on body weight and testicular weight of mice. A Body weight of normal mice and HC injured mice. B Testicular weight of normal mice and HC injured mice. Values represent the mean ± SD of independent experiments. Statistical significance was determined with one-way ANOVA followed by Dunnett’s test; n = 10
Discussion
For exploring the new side reaction of long-term administration of HC the pathological section of mouse testis was used to qualitatively visualize the damage of the testicular tissue, and to quantitatively calculate the number of spermatogonia, spermatocytes and the cells of the last stage of sperm formation in three spermatogenic phases. To get insight into the potential mechanism of the damage of the testicular tissue induced by HC the concentration of IL-2, IL-6 and TNF-α in both blood and testis of the long-term HC treated mice were determined by ELISA experiments. The results made these strategies to be of reference.
On the surface of normal seminiferous tubule, there were mature elongated spermatids distributed in pairs, and elongated spermatids close to maturity distributed alone, while the round spermatids at the developmental stage distributed under the lumen. On the the surface of abnormal seminiferous tubule of HC-damaged testis of the long-term HC treated mice, there were no mature elongated spermatids distributed in pairs, and no elongated spermatids close to maturity distributed alone, as well as no round spermatids at the developmental stage distributed under the lumen. Comparing with normal seminiferous tubules, the basement membrane boundary of HC-damaged seminiferous tubules became blurred, the villi and branches in the lumen became fewer and more disorderly, and the number of sperm was significantly decreased. Clinically, to avoid long-term administration of HC is critical for preventing the fertility ability of male from damage. These observations obviously suggested long-term administration of HC may damage the testicular tissue of juvenile mice. These findings emphasize that to avoid the damage of testicular function the reasonable administration of HC is of clinical importance for male adolescent patients.
The process of producing mature sperm in the seminiferous tubule was divided into the proliferative phase, the meiotic phase and the terminal phase of differentiation. Based on the morphology of spermatogonia and pathological sections of seminiferous tubule the number of spermatogonia in the three sphases was calculated. The data showed that by significantly decreasing the number of spermatogonia (compared with normal mice, P < 0.01), HC damaged the testicular function, thereby affected the reproductive function of male juvenile mice. These findings emphasize that to avoid the damage of testicular function the reasonable administration of HC is of clinical importance for male adolescent patients.
The spermatocytes were the key cells to germ cells. We explored that long-term administration of HC selectively decreased the number of spermatocytes in the proliferative phase (compared with normal mice, P < 0.01), but not in the meiotic phase and the terminal phase of differentiation (compared with sham mice, P > 0.05). These findings emphasize that to avoid the selective decrease of the number of spermatocytes in the proliferative phase the reasonable administration of HC is of clinical importance for male adolescent patients.
The number of spermatid mature spermatozoa cells could be used to judge the degree of testicular damage. We explored that long-term administration of HC may decrease the number of spermatid mature spermatozoa cells in the proliferative phase, the meiotic phase and the terminal phase of differentiation. This meant that long-term administration of HC damaged testis of juvenile mice. Therefore, to maintain testicular health the reasonable administration of HC is of clinical importance for male adolescent patients.
The expression level of IL-2, IL-6 and TNF-ɑ could be used to explore the molecular mechanism of testicular damage for male juvenile mice. We explored that long-term administration of HC simultaneously down-regulated the expression of IL-2, IL-6 and TNF-ɑ in both of the blood and the testicular tissue of juvenile mice. These mean that the expression levels of IL-2, IL-6 and TNF-ɑ in the blood was consistent with the expression levels of IL-2, IL-6 and TNF-ɑ in the testicular tissue.
Accordingly, IL-2, IL-6 and TNF-ɑ in the testicular tissue could be consisted as the molecular mechanism for the injury of the testicular tissue induced by long-term administration of HC, as well as IL-2, IL-6 and TNF-ɑ in the testicular tissue should be responsible for the alteration of the process and the sperm functionality of the male juvenile mice treated by HC.
Furthermore, IL-2, IL-6 and TNF-ɑ in the blood could be consisted as the molecular mechanism for the injury of the testicular tissue induced by long-term administration of HC, as well as IL-2, IL-6 and TNF-ɑ in the testicular tissue should be responsible for the alteration of the process and the sperm functionality of the male juvenile mice treated by HC.
Therefore, to avoid the risk of testicular injury male adolescent patients of long-term treated with HC should receive blood tests regularly to monitor the serum level of IL-2, IL-6 and TNF-ɑ. Besides, to maintain testicular health the reasonable administration of HC is of clinical importance for male adolescent patients as well.
In order to know long-term administration of HC whether or not affect the body weight and the testicular weight, the body weight and the testicular weight of the mice orally administered by HC for 16 days were compared with the body weight and the testicular weight of normal mice. We explored that long-term administration of HC did not affect the body weight and the testicular weight of the male juvenile mice (P > 0.05). This finding suggested that long-term administration of HC should not affect the body weight and the testicular weight of male adolescent patients.
Conclusions
HC is an important regulatory hormone and is widely used to treat a series of diseases clinically. Though HC may induce a number of side reactions, long-term administration of HC injuring testicular tissue is a newly founded side reaction. Thus avoiding testicular damage is crucial for male adolescent patients having long-term administration of HC, and this finding is of importance for improving the clinical applications of HC. The simultaneously down-regulate the expression of IL-2, IL-6 and TNF-ɑ in the blood and in the testicular tissue of the male juvenile mice meant that serum concentration of IL-2, IL-6 and TNF-ɑ reflected testicular concentration of IL-2, IL-6 and TNF-ɑ. Therefore, monitoring serum concentration of IL-2, IL-6 and TNF-ɑ was the same as monitoring testicular concentration of IL-2, IL-6 and TNF-ɑ for the male juvenile mice. Thus for the male juvenile mice the decrease of testicular concentration of IL-2, IL-6 and TNF-ɑ was consisted as the interleukins of injuring the testicular tissue induced by long-term administration of HC, while the decrease of the concentration of IL-2, IL-6 and TNF-ɑ in the blood could be used as the biomarker to reflect the progression of testicular injury induced by long-term administration of HC. Therefore, to avoid the risk of testicular injury male adolescent patients long-term treated with HC should receive blood test regularly for monitoring the concentration of IL-2, IL-6 and TNF-ɑ.
Acknowledgements
We thank all authors for their contributions and support for this article.
Author contributions
Xiaoyi, Zhang: review & editing, investigation, formal analysis, methodology, and data curation. Junhua zhou: writing-original draft, investigation, visualization, formal analysis, methodology, and data curation. Yifan Yang: visualization and investigation. Yaonan Wang: methodology, software, and visualization. Shurui Zhao: methodology, software, and investigation. Jianhui Wu, Ming Zhao and Shiqi Peng: writing-review & editing, conceptualization, supervision, funding acquisition, conceptualization, and project administration. The authors have read and approved the final manuscript.
Funding
The research was supported by a grant from the Chinese Institutes for Medical Research, Beijing, CX25YQ02.
Data availability
No datasets were generated or analysed during the current study.
Declarations
Ethical approval
The animal experiments in this study were reviewed and approved by Ethics Committee of Capital Medical University (Ethical Approval Number: AEEI-2021-202).
Consent for publication
All authors approved the final manuscript and the submission to this journal.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Xiaoyi Zhang, Junhua Zhou and Yifan Yang contributed equally to this work.
Contributor Information
Jianhui Wu, Email: wujianhui01@126.com.
Ming Zhao, Email: zhaomingccmu@ccmu.edu.cn.
Shiqi Peng, Email: sqpeng@bjmu.edu.cn.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
No datasets were generated or analysed during the current study.