A Docosahexaenoic Acid Derivative (N-Benzyl Docosahexaenamide) as a Potential Therapeutic Candidate for Treatment of Ovarian Injury in the Mouse Model

Abstract

Commonly used clinical chemotherapy drugs, such as cyclophosphamide (CTX), may cause injury to the ovaries. Hormone therapies can reduce the ovarian injury risk; however, they do not achieve the desired effect and have obvious side effects. Therefore, it is necessary to find a potential therapeutic candidate for ovarian injury after chemotherapy. N-Benzyl docosahexaenamide (NB-DHA) is a docosahexaenoic acid derivative. It was recently identified as the specific macamide with a high degree of unsaturation in maca (Lepidium meyenii). In this study, the purified NB-DHA was administered intragastrically to the mice with CTX-induced ovarian injury at three dose levels. Blood and tissue samples were collected to assess the regulation of NB-DHA on ovarian function. The results indicated that NB-DHA was effective in improving the disorder of estrous cycle, and the CTX+NB-H group can be recovered to normal levels. NB-DHA also significantly increased the number of primordial follicles, especially in the CTX+NB-M and CTX+NB-H groups. Follicle-stimulating hormone and luteinizing hormone levels in all treatment groups and estradiol levels in the CTX+NB-H group returned to normal. mRNA expression of ovarian development-related genes was positive regulated. The proportion of granulosa cell apoptosis decreased significantly, especially in the CTX+NB-H group. The expression of anti-Müllerian hormone and follicle-stimulating hormone receptor significantly increased in ovarian tissues after NB-DHA treatment. NB-DHA may be a promising agent for treating ovarian injury.

Keywords: cyclophosphamide; docosahexaenoic acids; granulosa cells; macamide; ovary.

Figure 1

Chromatograms and structural formulas of DHA-EE and NB-DHA. (A) Chromatograms of synthetic DHA-EE material and purified DHA-EE sample collected using a semipreparative HPLC system (26.5–30.5 min). (B) Chromatograms of synthetic NB-DHA material and purified NB-DHA sample collected using a semipreparative HPLC system (26.0–29.0 min). Structural formulas of DHA-EE (C) and NB-DHA (D).

Figure 2

Body weight, ovarian weight, frequency of occurrence, and estrous cycle. Weight Change/% (A) and ovarian weight (B) measured on days 1, 4, 7, 10, 13, 16, 19, and 22 after administration. Initial average weight of mice was set as 100%. Effect of DHA-EE and NB-DHA on estrous cycles. Frequency of occurrence of cycle stages during the 21 days (n = 6) (C) and estrous cycle regularity (D). Values in all figures are expressed as the mean ± SEM (x ± sem, n = 6), ** p < 0.01, *** p < 0.005 (the same below).

Figure 3

Effect of DHA-EE and NB-DHA on the development of follicles. Follicles after H&E staining (A). Magnification 100× and 400×. Scale bar: 250 and 50 μm. Number of different follicles: primordial follicles (B), primary follicles (C), secondary follicles (D), and atretic follicles (E). *** p < 0.005.

Figure 4

Effect of model and treatment on sex hormone levels and mRNA expression in mice. Sex hormone levels of E2 (A), AMH (B), FSH (C), and LH (D). mRNA expression levels of FOXL2 (E), GDF9 (F), LIF (G), OCT4 (H), and SCF (I) in mouse ovarian tissues, as determined by real-time PCR. * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 5

TUNEL and immunohistochemical analysis. Apoptosis of granulosa cells in ovarian tissues was measured via TUNEL assay (A). Green fluorescence: apoptotic cells; blue fluorescence: nucleus; magnification: 400×. The expression of AMH and FSHR in ovarian tissues were measured by immunohistochemical analysis (B). Blue: nucleus; brown: cells expressing AMH and FSHR in the cytoplasm; scale bar: 50 μm; magnification: 400×. TUNEL-positive cell ratios in the treatment groups were analyzed according to the number of TUNEL-positive granular cells among all granulosa cells in the follicle (C). Scoring of staining results (D,E). ** p < 0.01, *** p < 0.005.