PQQ Dietary Supplementation Prevents Alkylating Agent-Induced Ovarian Dysfunction in Mice

Abstract

Alkylating agents (AAs) that are commonly used for cancer therapy cause great damage to the ovary. Pyrroloquinoline-quinine (PQQ), which was initially identified as a redox cofactor for bacterial dehydrogenases, has been demonstrated to benefit the fertility of females. The aim of this study was to investigate whether PQQ dietary supplementation plays a protective role against alkylating agent-induced ovarian dysfunction. A single dose of busulphan (20 mg/kg) and cyclophosphamide (CTX, 120 mg/kg) were used to establish a mouse model of ovarian dysfunction. Feed containing PQQNa₂ (5 mg/kg) was provided starting 1 week before the establishment of the mouse model until the date of sacrifice. One month later, estrous cycle period of mice were examined and recorded for consecutive 30 days. Three months later, some mice were mated with fertile male mice for fertility test. The remaining mice were sacrificed to collect serum samples and ovaries. One day before sacrifice, some mice received a single injection of BrdU to label proliferating cells. Serum samples were used for test hormonal levels. Ovaries were weighted and used to detect follicle counts, cell proliferation, cell apoptosis and cell senescence. In addition, the levels of inflammation, oxidative damage and Pgc1α expression were detected in ovaries. Results showed that PQQ treatment increased the ovarian weight and size, partially normalized the disrupted estrous cycle period and prevented the loss of follicles of mice treated with AAs. More importantly, we found that PQQ treatment significantly increased the pregnancy rate and litter size per delivery of mice treated with AAs. The protective effects of PQQ appeared to be directly mediated by promoting cell proliferation of granulosa, and inhibiting cell apoptosis of granulosa and cell senescence of ovarian stromal cells. The underlying mechanisms may attribute to the anti-oxidative stress, anti-inflammation and pro-mitochondria biogenesis effects of PQQ.Our study highlights the therapeutic potential of PQQ against ovarian dysfunction caused by alkylating agents.

Keywords: alkylating agents; ovarian aging; ovarian dysfunction; protection; pyrroloquinoline quinine.

Figure 1

PQQ treatment improved the fertility and ovarian function of female mice treated with CTX in combination with BUL (CTX/BUL). (A) Record of estrous cycles of mice for each day, n = 6 mice in each group. (B) Estrous cycle period distribution for 30 consecutive days, n = 6 mice in each group. (C) Fertility test of female mice, n = 7 mice in each group. (pregnancy mice/total mice). (D) Days from mating to delivery, n = 7 mice in each group. ELISA analysis of serum (E) AMH, (F) FSH and (G) E2 levels, n = 4 samples in group Con, n=7 samples in group O, and n=5 samples in group OP. D, diestrus; M, metestrus; E, estrus; P, proestrus; AMH, anti-Mullerian hormone; FSH, follicle-stimulating hormone; E2, estradiol; PQQ, pyrroloquinoline; CTX, cyclophosphamide; BUL, busulfan. Compared with Con: *P < 0.05; **P < 0.01; ***P < 0.001; compared with O: ^#P < 0.05; ^###P < 0.001.

Figure 2

PQQ treatment partially prevented follicles loss in the ovaries of female mice treated with CTX/BUL. (A) Ovarian size, n = 6 mice in each group. (C) Representative photos of ovarian sections stained with HE, n = 3 mice. Black arrow indicates the primordial follicle; red triangle indicates the degenerative follicle. (B) Ovarian weight, n=6 mice. (D) Follicle counts in each stage, Pr, primordial follicle; Pm, primary follicles; Se, secondary follicle; Ma, mature follicle; De, degenerative follicle; PQQ, pyrroloquinoline; CTX, cyclophosphamide; BUL, busulfan. Compared with Con: *P < 0.05; **P < 0.01; ***P < 0.001; compared with O: ^#P < 0.05.

Figure 3

PQQ treatment promoted ovarian cell proliferation and inhibited ovarian cell apoptosis and senescence in the female mice treated with CTX/BUL. (A) Representative photos of ovarian sections stained with anti-BrdU antibody, n = 3 mice in each group. (B) Representative photos of TUNEL assay on ovarian sections, n = 3 mice in each group. (C) Representative photos of ovarian sections stained with anti- β-gal antibody, n = 3 mice in each group. q-PCR analysis of ovarian extracts to detect the expression of the (D) Bmi1 gene, n = 6 mice in each group, (E) p21 gene, n = 6 mice in each group, (F) p27 gene, n = 6 mice in each group, (G) p53 gene, n = 6 mice in each group, (H) Bax gene, n = 6 mice in each group, (I) Noxa gene, n = 6 mice in each group and (J) puma gene, n = 6 mice in each group. PQQ, pyrroloquinoline; CTX, cyclophosphamide; BUL, busulfan. Compared with Con: *P < 0.05; **P < 0.01; ***P < 0.001; compared with O: ^#P < 0.05; ^##P < 0.01.

Figure 4

PQQ treatment decreased inflammation in the ovaries of female mice treated with CTX/BUL. q-PCR analysis of ovarian extracts to detect the expression of the (A) IL-1α gene, n = 6 mice in each group, (B) IL-1β gene, n = 6 mice in each group, (C) IL-6 gene, n = 6 mice in each group, and (D) TNF α gene, n = 6 mice in each group. (E) Representative photos of ovarian sections stained with anti-p65 antibody, n = 3 mice in each group. PQQ, pyrroloquinoline; CTX, cyclophosphamide; BUL, busulfan. Compared with Con: *P < 0.05; **P < 0.01; ***P < 0.001; compared with O: ^#P < 0.05; ^###P < 0.001.

Figure 5

PQQ treatment decreased oxidative damage and increased the expression of Pgc1α in the ovaries of female mice treated with CTX/BUL. (A) Detection of MDA concentration from ovarian extracts, n = 6 mice in each group. (B) Representative photos of ovarian sections stained with anti-8-OHdG antibody, n = 3 mice in each group. (C) q-PCR analysis of ovarian extracts to detect the expression of the Pgc1α gene, n = 6 mice in each group. PQQ, pyrroloquinoline; CTX, cyclophosphamide; BUL, busulfan. Compared with Con: *P < 0.05; **P < 0.01; ***P < 0.001; compared with O: ^#P < 0.05; ^##P < 0.05.