Effect of Curcuma longa maceration treatment on ovarian follicular development, serum oestradiol, uterine growth and vascularisation in female albino rats
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Introduction
Curcuma longa, a plant producing turmeric, has long been recognised for its medicinal properties. The benefits of C. longa’s rhizome to health are largely attributed to its most prominent biologically-active compound, curcumin, found in turmeric (12). The multiple pharmacological benefits of herbs including curcumin are seen in antioxidant, anti-inflammatory, antimicrobial and anti-cancer activity (8, 10, 16). The beneficial properties of curcumin were further demonstrated in female animal studies. Curcumin treatments were reported to improve the ovulation and corpus luteum of rats with polycystic ovary syndrome by inhibiting the expression of tumour necrosis factor alpha (TNF-α), interleukin 6, and C-reactive protein. Curcuma longa and curcumin could inhibit the production of TNF-α and prostaglandin E2 as well as suppress the production of vascular endothelial growth factor (VEGF) in ovarian cancer. Curcumin also attenuated ovarian failure and improved embryonic development in aging mice (5, 13).
However, studies on the effects of C. longa and its active components on the reproductive function of female animals are limited. Administration of ethanol extract of C. longa in female rats was reported to increase the weight of the ovary, uterus and body (1). Another study reported that dietary C. longa in female rabbits improved folliculogenesis and ovarian viability by increasing progesterone synthesis and secretion and modulating luteinising hormone receptors and the release of leptin by the ovary (7). Phytooestrogenic effects of curcumin have been described, but there are very few studies investigating the action of C. longa on the regulation of endocrine functions in female reproduction and how it affects the reproductive organs. Therefore, the objective of the study was to evaluate the improving effect of C. longa on the uterus, uterine and ovarian development and vascularisation, and hormone synthesis and secretions in female Sprague Dawley rats.
Material and Methods
Preparation of macerated
C. longa. A 100 g mass of fresh C. longa was coarsely chopped and then added into 100 mL of boiling water. The resulting maceration was chilled at room temperature for 1 h and filtered using 100 μm wire mesh to be a stock solution. By diluting this with mineral water, 1% and 5% drinking water solutions were prepared. The stock solutions were prepared freshly every three days and kept in a refrigerator at 4°C.
Experimental procedure
The experimental rats were divided into three treatment groups, each group consisting of five females (according to the Federer formula). The Control group was only given mineral water without C. longa content, the Cur-Low group was given mineral water containing 1% C. longa maceration, and the Cur-High group was given mineral water containing 5% C. longa maceration.
Follicle stimulating hormone, oestradiol and progesterone assays
Intracardiac blood was collected into centrifuge tubes and serum was prepared by centrifugation (1,500 × g for 20 min at 4°C). Serum was then stored at –20°C until it was assayed. The hormone, oestradiol and progesterone concentrations were measured using a Rat FSH (Follicle Stimulating Hormone) ELISA Kit, Rat/Porcine E2(Estradiol) ELISA Kit and QuicKey Pro Rat Pg(Progesterone) ELISA Kit (all products of Elabscience, Houston, TX, USA) according to the manufacturer’s instructions.
Uterine and ovarian relative weight
The relative weight of the uterus and the ovary was determined by dividing each organ’s weight by the rat’s body weight.
Macroscopic examination of the uterus
Uterus images were captured during euthanasia to represent the organ under ideal vascular conditions. The diameter and length of the left and right uterine cornua and the proportion of uterine blood vessels were measured using ImageJ image analysis (23).
Uterine gland measurement and ovarian follicle counting
Formalin-fixed blocks of the ovary and uterus tissue embedded in paraffin were cut into sections of 4 μm using a Leica RM2135 Microtome (Leica Biosystems, Nussloch, Germany). Tissue sections were then placed onto a glass slide, deparaffinised in xylene, rehydrated in graded alcohols, stained with haematoxylin and eosin (H&E) and sealed with Entellan (Merck, Darmstadt, Germany). All experimental groups’ tissue samples were evaluated on H&E-stained slides. The diameter of the uterine gland, the number of primary, secondary, tertiary and Graafian follicles, and the number of corpora lutea were determined using image analysis methods in ImageJ.
Immunohistochemical staining of ovarian VEGF
Immunoperoxidase staining was performed to investigate changes in VEGF. Glass slides were coated with poly-l-lysine (Biogear, Indonesia). Then, slide sections of the ovary were dewaxed, rehydrated and submitted for endogenous peroxidase quenching. Antigen epitope retrieval was enhanced with heat-induced epitope retrieval solution (ScyTek, Logan, UT, USA) for 5 min at 121°C. Tissue slide sections were washed with 10 mM PBS with pH of 7.4 for 5 min, then incubated with a mouse monoclonal antibody to VEGF at a dilution of 1:50 (C-1 clone raised against amino acids of VEGF-A of human origin, sc-7269; Santa Cruz Biotechnology, Dallas, TX, USA), immersed in wash buffer for 5 min and visualised with EnVision FLEX polymer (Dako Omnis, Agilent, Santa Clara, CA, USA) for 20 min.
Statistical analyses
Analyses were performed using R software 3.5.1 version (21). The data expressed as mean ± standard deviation were analysed by one-way analysis of variance followed by Duncan’s post-hoc test. A P-value < 0.05 was considered statistically significant.
Results
The relative weights of the uterus and of the ovary of the experimental rats are shown in Fig. 1.
Fig. 1.
The uterine (A) and ovary (B) relative weight of experimental Sprague Dawley rats after 20 days’ treatment with Curcuma longa maceration at doses of 0% (Control), 1% (Cur-Low) or 5% (Cur-High). ANOVA – one-way analysis of variance; * – significant difference compared to the control group (P-value < 0.05); ns – non significant
The relative weight of the uterus was significantly higher in rats treated with C. longa maceration at doses of 1% (Cur-Low) and 5% (Cur-High) (P-value < 0.05) compared to that in control rats without administration of C. longa maceration. However, there was no significant difference in the relative weights of the uterus between the Cur-Low and Cur-High groups. The variation of the relative weights of the uterus and ovary was quite high (Fig. 1). The results also show significantly larger uterine cornua diameters in experimental rats compared to the control rats (P-value < 0.05) (Fig. 2).
Fig. 2.
Diameter (A), length (B), and vascularisation (C) of uterus in experimental Sprague Dawley rats after 20 days’ treatment with Curcuma longa maceration at doses of 0% (Control), 1% (Cur-Low) or 5% (Cur-High). ANOVA – one-way analysis of variance; NS/ns – non significant
The macroscopic evaluation of uterine vascularisation in the experimental Sprague Dawley rats treated with different doses of C. longa maceration is presented in Fig. 3. Stronger uterine vascularisation was observed in the uteri of experimental rats treated with 1% (Cur-Low) and 5% (Cur-High) C. longa maceration compared to the uteri of control rats. In accordance with the macroscopic observations, histological evaluation of the uterus in the experimental rats treated with C. longa maceration showed an improvement in gland activity, which was indicated by the larger sizes of the uterine glands. The size of the lumen of the uterine glands in the experimental rats treated with C. longa maceration at both doses was observed to be larger than that in the Control group (P-value < 0.05) (Fig. 4). Histological examination was also performed to count the number of ovarian follicles. The results showed that treatment with 1% and 5% C. longa maceration significantly increased the number of tertiary follicles and corpora lutea in the experimental rats compared to the control animals (P-value < 0.05) (Fig. 5). Vascular endothelial growth factor expression in the interstitial tissue of the ovary indicates the stimulated progression of neovascularisation, which is important for ovarian function and particularly for follicular development. Expression of VEGF revealed by immunostaining in both the Cur-Low and Cur-High groups was statistically significantly higher than that in the Control group (Fig. 6).
Fig. 3.
Gross pathology of the uterus and its vascularisation in experimental Sprague Dawley rats after 20 days’ treatment with Curcuma longa maceration at doses of 0% (A – Control), 1% (B – Cur-Low) or 5% (C – Cur-High)
Fig. 4.
Histopathological visualisation showing the effect of 20 days’ Curcuma longa maceration administration on the uterine gland of experimental Sprague Dawley rats. The maceration was administered at doses of 0% (A – Control), 1% (B – Cur-Low) or 5% (C – Cur-High)
Fig. 5.
Histopathological visualisation showing the effect of 20 days’ Curcuma longa maceration administration on the follicles of experimental Sprague Dawley rats. The maceration was administered at doses of 0% (A – Control), 1% (B – Cur-Low) or 5% (C – Cur-High)
Fig. 6.
Immunohistochemical staining showing the effect of 20 days’ Curcuma longa maceration administration on the expression of ovarian vascular endothelial growth factor by experimental Sprague Dawley rats. The maceration was administered at doses of 0% (A – Control), 1% (B – Cur-Low) or 5% (C – Cur-High)
The overall serum concentrations of FSH in experimental rats treated with C. longa at a dose of 1% (the Cur-Low group) were significantly lower than those in the control rats without C. longa treatment, while in the experimental rats treated with C. longa maceration at a dose of 5% (the Cur-High group), only three out of five serum samples’ concentrations were significantly lower (Fig. 7A). Serum oestradiol concentrations were increased in the experimental rats treated with 1% and 5% C. longa maceration, with four out of five samples from the Cur-Low group showing a significant difference (P-value < 0.05) compared to the control rats (Fig. 7B). There was no significant difference observed in the serum progesterone concentration between control rats and rats treated with C. longa maceration at doses of 1% and 5%, and only three out of five samples’ concentrations from the Cur-Low group and two out of five samples’ concentrations from the Cur-High group were higher than those in the control group (Fig. 7C).
Fig. 7.
Serum follicle-stimulating hormone (FSH) (A), oestradiol (B) and progesterone (C) concentrations in experimental female Sprague Dawley rats after 20 days’ treatment with Curcuma longa maceration at doses of 0% (Control), 1% (Cur-Low) or 5% (Cur-High). ANOVA – one-way analysis of variance; * – significant difference compared to the control group (P-value < 0.05); ns – non significant
Discussion
The administration of C. longa maceration for 20 days to mature female Sprague Dawley rats in their dioestrous phase showed great potential as an alternative method for improving the gonadal growth and development of female animals. The findings of the higher relative weight of the ovary and the significantly higher number of tertiary follicles and corpora lutea point to these reproductive health benefits being imparted by C. longa administration. The extract of this plant may reduce ovarian cell death and boost folliculogenesis through the anti-oxidative properties of curcumin, the primary active component of turmeric. In studies conducted previously to evaluate the C. longa effect on female rabbit reproduction, dietary turmeric increased the number of primary follicles, as well as the diameter of primary, secondary and tertiary follicles, which suggests that turmeric promotes the production of primary ovarian follicles and stimulates follicle growth throughout folliculogenesis (2, 6).
In addition to the direct mechanism of action by which C. longa produces its pharmacological effect of strengthening the development of the ovary and its function, another suggested pathway along which the reproductive health–enhancing effect of C. longa is exerted is the augmentation of VEGF expression. Rats treated with C. longa maceration had higher expression of VEGF in the ovary. Ovarian function is dependent on the establishment and continual remodelling of a complex vascular system, and VEGF is an important factor in this mechanism. The growth factor can increase vascular permeability and stimulate angiogenesis, and it particularly functions as a stimulator for endothelial proliferation and migration (3, 20).
The follicle-stimulating hormone serum concentration indicates how much FSH is circulating in the blood. In this study, most rats given C. longa treatment had lower serum FSH concentrations than control rats. These data indicated that FSH was metabolised for reproductive activity, and particularly to stimulate the growth and development of follicles in the ovary; it was observed to interact with membrane receptors in the ovary to stimulate the signalling pathway of folliculogenesis during follicle growth and development (25, 27).
Oestradiol was more highly concentrated with C. longa maceration treatment, and the greater concentration consequently raised the relative weight and the size of the uteri over those of control rats. In previous studies, the administration of oestradiol to rodents induced uterine growth particularly in the endometrium, and that growth was affected by the bioavailability of oestradiol in serum (17, 18). In our present study, the level of endogenous oestradiol was exceptionally elevated. Curcumin’s phytooestrogenic properties and stimulatory effect upon oestradiol secretion contributed to the increased relative weight of the uterus. Furthermore, the upregulation of oestradiol caused by C. longa administration also improved the vascularisation of the uterine cornua. Stronger vascularisation also directly contributes to the higher uterine relative weight and is one of the important indications of better-developed uteri (15, 19).
Elevated oestradiol and progesterone concentrations further improve the development of the uterus and particularly of the uterine glands. Rats supplemented with C. longa maceration benefitted from better gland development. It was reported in previous studies that uterine function is primarily regulated by ovarian oestrogen and progesterone (4, 14). These hormones influenced the development of luminal and glandular epithelia and induced the stroma to express genes that are important for uterine receptivity and blastocyst implantation, including cytokines, growth factors and lipid mediators (9, 11). As described in the previous study by Kelleher et al. (14), the uterine gland is essential for fertility and pregnancy, as its secretions and products affected embryo survival and implantation in humans and rodents, which also mediated stromal cell decidualisation and placental development in mice.
The 20-day experiment was performed in the dioestrous phase, which has the longest duration but which also varied among individuals (around 48–72 h in rats), and this variability implied that the experimental rats were not in exactly the same stage of the oestrous cycle. Nevertheless, exceptional effects of the supplementation of C. longa maceration on sex steroid hormone concentrations and gonad development were still highly manifest in the parameters measured in the present study. Therefore, C. longa has the potential to be developed and used to improve reproduction performance and produce better quality and performance of the offspring (22, 24).
Conclusion
Application of Curcuma longa maceration in female albino rats made their reproductive organs better able to support gestation by improving their morphological profile, quality and functions. Curcuma longa maceration is also a potential supplement to refine the signalling pathway of the reproductive system to promote a healthy prenatal environment for the optimum growth and development of embryos and foetuses of animals.
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