Contributed equally
Curcumin, a type of natural active ingredient, is derived from rhizoma of Curcuma, which possesses antioxidant, antitumorigenic and anti-inflammatory activities. The present study aimed to investigate whether treatment with curcumin reduced high-fat diet (HFD)-induced spermatogenesis dysfunction. Sprague-Dawley rats fed a HFD were treated with or without curcumin for 8 weeks. The testis/body weight, histological analysis and serum hormone levels were used to evaluate the effects of curcumin treatment on spermatogenesis dysfunction induced by the HFD. In addition, the expression levels of apoptosis associated proteins, Fas, B-cell lymphoma (Bcl)-xl, Bcl-associated X protein (Bax) and cleaved-caspase 3, were determined in the testis. The results of the present study suggested that curcumin treatment attenuated decreased testis/body weight and abnormal hormone levels. Morphological changes induced by a HFD were characterized as atrophied seminiferous tubules, decreased spermatogenetic cells and interstitial cells were improved by curcumin treatment. In addition, curcumin treatment reduced apoptosis in the testis, and decreased expression of Fas, Bax and cleaved-caspase 3, as well as increased expression of Bcl-xl. In conclusion, the present study revealed that curcumin treatment reduced HFD-induced spermatogenesis dysfunction in male rats.
Infertility is defined as when couples who have an active sex life without using protective measures for >1 year, fail to get pregnant, which affects 10–15% (
Curcumin, a type of natural active ingredient derived from rhizoma of Curcuma, has protective effects in a series of diseases, including cardiovascular disease (
A total of 30 male Sprague-Dawley rats (permit number, 42000500002649), weighing 200–250 g were used in the present study. All animals experiments performed in the present study were approved by the Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University (Wuhan, China). The animals were allowed free access to food and water at all times and were maintained on a 12 h light/dark cycle at a controlled temperature (20–25°C) and humidity (50±5%), which was also pathogen-free. The composition of the HFD is shown in
The collected testis tissues were fixed with 4% paraformaldehyde, dehydrated and embedded in paraffin (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The testis tissue sections (5
Blood samples were obtained from the abdominal aorta. Following centrifugation (1,506 × g) for 10 min at 4°C, the sera were obtained for further detection. Serum levels of estradiol (E2), testosterone (T), follicle stimulating hormone (FSH), luteinizing hormone (LH) and leptin were measured using kits, according to the manufacturer's protocol (Elabscience Biotechnology Co., Ltd., Wuhan, China).
The protein expression levels of Fas, Bcl-xl and leptin receptors were tested by immunohistochemistry. The sections were deparaffinized and were subsequently boiled for 15 min in sodium citrate buffer for antigen retrieval. Following elimination of internal peroxidase activity, the sections were incubated with rabbit anti-Fas (1:50; cat no. BA0408), rabbit anti-leptin-receptor (1:50; cat no. BA1233; Wuhan Boster Biological Technology, Ltd., Wuhan, China) and rabbit anti-Bcl-xl (1:100; cat. no. 10783-AP; Proteintech Group, Inc., Chicago, IL, USA) antibodies at 4°C overnight. The tissue sections were exposed to biotinylated sheep anti-rabbit immunoglobulin G solution (Proteintech Group, Inc.) at 37°C for 30 min and were subsequently incubated with horseradish peroxidase-labeled streptavidin (Proteintech Group, Inc.) at 37°C for 30 min. Finally, the tissue sections were observed under light microscopy (Nikon E100; Nikon), and the photomicrographs were obtained by Photo Imaging System (Canon 600D; Canon). In each group 30 fields (5 fields/rat) in 6 rat testis were randomly selected. Positive expression was assessed using Image-Pro Plus 6.0 and a mean of the integrated optical density was obtained.
Tissue sections were processed, according to the manufacturer's protocol for the TUNEL kit (Roche Applied Science, Indianapolis, IN, USA). Positively labeled nuclei were stained a brown color, while negatively labeled nuclei were blue. A total of 30 fields were randomly selected in each group (5 fields/rat) and 100 cells were counted in each field under a microscope (Nikon E100; Nikon), and the number of positive cells was recorded. The apoptosis index (number of apoptotic cells in each field/100) was computed for each field.
Lysis buffer (720
All statistical analyses were performed with SPSS 19.0 (IBS SPSS, Chicago, IL, USA). All data are expressed as the mean ± standard error of the mean. Multiple group comparison was performed by analysis of variance, followed by the post-hoc least significant difference test assuming equal variances; otherwise Tamhane's T2 post-hoc test. All statistical analyses were two-sided. P<0.05 was considered to indicate a statistically significant difference.
Following a HFD for 8 weeks, the testis/body weight was reduced and treatment with curcumin increased the testis/body weight (
Rats fed with an HFD exhibited abnormal serum hormone levels, manifested as reduced serum levels of T, FSH and LH, and increased serum levels of leptin and E2. Serum hormone levels were observed to be restored following treatment with curcumin (
A HFD resulted in more apoptotic cells in the testis, characterized by elevated expression levels of Fas, Bax and cleaved-caspase 3, and reduced expression of Bcl-xl. Following treatment with curcumin, these changes were attenuated (
In order to investigate the effects of curcumin on male spermatogenesis function, the present study subjected the rats to a HFD to induce spermatogenesis dysfunction. The present results revealed that spermatogenesis function was disturbed in rats fed with a HFD, characterized as decreased testis/body weight, atrophied seminiferous tubules, reduced spermatogenetic and interstitial cells, and abnormal hormone levels, which is consistent with a previous study (
Curcumin has been demonstrated to have a series of pharmacological actions, including antioxidant, antimutative, anti-inflammatory and antitumorigenic actions (
The balance between cell proliferation and cell death is important in spermatogenesis function. As a type of cell death, apoptosis of testicular germ cells was an important physiological mechanism in regulating the germ cell population (
Leptin, also synthesized by seminiferous tubules (
In conclusion, the present study revealed that curcumin reduced HFD-induced spermatogenesis dysfunction and apoptosis. Curcumin may be a potential novel therapeutic medicine for male patients suffering from obesity-induced infertility.
The present study was partially supported by the Key Research Project of the Ministry of Public Security (no. 2010 ZDYJHBST007).
Oral treatment with curcumin prevents HFD-induced male spermatogenesis dysfunction. (A) Hematoxylin and eosin staining of the testis in the three groups (n=6; magnification, ×100 and ×400; scale bar, 100 and 20
Curcumin treatment improves defective serum hormone levels of (A) T, (B) E2, (C) FSH and (D) LH induced by HFD. (n=8; *P<0.05, HFD vs. control group; #P<0.05, curcumin treatment group vs. HFD group). FSH, follicle-stimulating hormone; LH, luteinizing hormone; T, testosterone; E2, estradiol; HFD, high-fat diet.
Curcumin supplementation attenuates apoptosis in the testis. (A) Immunohistochemical staining for the protein expression levels of Bcl-xl and Fas, and TUNEL for the apoptotic cells in the testis (n=6; magnification ×100 and ×400; scale bar 100 and 20
Curcumin treatment reduces apoptosis-associated protein expression levels of Bax and cleaved-cas-3. (A) Western blotting analysis of the protein expression levels of Bax and cleaved-cas-3 was assessed in the testis tissues from the three groups (n=4). The fold-change in protein expression levels of (B) Bax and (C) cleaved-cas-3 was quantified relative to the expression of GAPDH. The data are expressed as the mean ± standard error of the mean (*P<0.05, HFD vs. control group; #P<0.05, curcumin vs. HFD group. HFD, high-fat diet; cas, caspase; Bax, B-cell lymphoma associated X protein.
Curcumin treatment decreases the expression of leptin receptors and reduces serum leptin level. (A) Immunohistochemical staining for the expression of leptin receptors in the testis (n=6; magnification ×100 and ×400; scale bar 100 and 20
Macronutrient composition of diets for rats.
Nutrient | Control
|
High-fat diet
| ||
---|---|---|---|---|
g, % | kJ, % | g, % | kJ, % | |
Protein | 20 | 19 | 20 | 14 |
Carbohydrate | 76 | 72 | 45 | 31 |
Saturated fat | 4 | 9 | 35 | 55 |
kJ/g | 17.5 | 24.1 |
Testicular weight and testicular/body weight of the three groups.
Group | n | Body weight (g) | Testicular weight (g) | Testicular/body weight (g/kg) |
---|---|---|---|---|
Control | 10 | 315.80±14.74 | 3.14±0.21 | 9.97±0.93 |
High-fat diet | 10 | 371.40±12.05 |
2.70±0.16 |
7.27±0.35 |
Curcumin | 10 | 353.20±5.76 | 2.90±0.12 |
8.21±0.38 |
The data are expressed as the mean ± standard error of the mean.
P<0.05 vs. control group;
P<0.05 vs. high-fat diet group.
Comparison of diameters of seminiferous tubules and the quantity of spermatogenetic cells and interstitial cells in the three groups.
Group | n | Seminiferous tubule diameter ( |
Quantity of spermatogenetic cells | Quantity of interstitial cells |
---|---|---|---|---|
Control | 6 | 395.08±19.91 | 25.40±1.14 | 8.16±0.29 |
High-fat diet | 6 | 273.70±16.55 |
14.80±1.79 |
4.16±0.12 |
Curcumin | 6 | 319.94±20.55 |
20.60±1.14 |
5.37±0.11 |
The data are expressed as the mean ± standard error of the mean.
P<0.05 vs. control group;
P<0.05 vs. HFD group.