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Effect of molybdenum on reproductive function of male mice treated with busulfan
Accepted Manuscript Effect of molybdenum on reproductive function of male mice treated with busulfan
Feng-Jun Liu, Wen-Ya Dong, Hao Zhao, Xin-Huai Shi, Yu-Ling Zhang PII:
S0093-691X(18)31087-2
DOI:
10.1016/j.theriogenology.2018.12.002
Reference:
THE 14794
To appear in:
Theriogenology
Received Date:
17 April 2018
Accepted Date:
01 December 2018
Please cite this article as: Feng-Jun Liu, Wen-Ya Dong, Hao Zhao, Xin-Huai Shi, Yu-Ling Zhang, Effect of molybdenum on reproductive function of male mice treated with busulfan, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.12.002
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Effect of molybdenum on reproductive function of male mice treated with busulfan
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Feng-Jun Liu*, Wen-Ya Dong, Hao Zhao, Xin-Huai Shi, Yu-Ling Zhang
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Animal College of Science and Technology, Henan University of Science and
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Technology, Luoyang 471003, PR China.
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Abstract
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Infertility is a serious public health problem worldwide. Molybdenum (Mo)
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plays an important role in maintaining normal metabolism. To explore the therapeutic
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efficacy of molybdenum (Mo) on male infertility, 90 mice were randomly divided
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into control, busulfan and busulfan+Mo groups. The male mice in the busulfan and
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busulfan+Mo groups were exposed to busulfan (20 mg/kg body weight) with a single
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intraperitoneal injection to establish the infertility model. The sterile mice were
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successfully obtained 30 days after busulfan exposure. Then, the male mice in the
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busulfan+Mo group were given drinking water containing 20 mg/L Mo continuously
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for 42 days. At 72 Day after treatment, 30 mice in the three groups were tested for
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various indices, and 60 mice were mated with females in spontaneous estrus. Mo
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significantly reversed the thinner seminiferous tubules and disappeared tubule and
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germ cells. Mo also normalized previously abnormal levels of testosterone, estradiol,
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luteinizing hormone, superoxide dismutase, lactate dehydrogenase, malondialdehyde.
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Furthermore, expression levels expression of Bax, Bcl-2, caspase-3 and caspase-9 * Corresponding author at: College of Animal Science and Technology, Henan University of Science and Technology, No.263 Kaiyuan Avenue, Luoyang, China, 471023. Email: [email protected] (Feng-jun Liu) 1
Revised
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returned to control levels; and finally, Mo-treated sterile mice obtained offspring with
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normal number and gender ratio. These results suggested that Mo at 20 mg/L had a
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significant therapeutic effect on reproductive dysfunction in sterile mice. Its
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mechanism could via repair of damaged testicular structures, regulation of abnormal
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reproductive hormone levels, decreased oxidative stress or and resistance to cell
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apoptosis. Mo may be a new candidate medicine for treatment of male infertility.
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Key- words: molybdenum; busulfan; infertility; testis; mouse
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1. Introduction
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Infertility is a serious health problem worldwide, and its incidence is increasing.
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Currently, the incidence of the infertility is approximately 24% in humans, with 18%
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in the men [1]. Infertility may be caused by a variety of factors, including disease,
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environmental pollution, endocrine disorders and reproductive system injuries [2-5].
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The testis is the only organ that generates sperm in the male reproductive system. Its
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normal structure and function are important for the maintenance of male reproduction
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[6]. Therefore, any disturbance to the testis may result in male infertility.
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Reproductive hormone alterations, apoptosis, oxidative damage and abnormal tissue
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structures in the testis and epididymis might be mechanisms for the degeneration or
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disappearance of male reproductive function [7-9]. The abnormal synthesis and
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metabolism of reproductive hormones, such as FSH (follicle stimulating hormone),
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luteinizing hormone (LH), testosterone (T) and estradiol (E2) could cause
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reproductive disorders [10-12]. Imbalances in redox states involving superoxide 2
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dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA)
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have been shown to mediate male infertility associated with many diseases [13, 14].
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Furthermore, the high expression of apoptotic factors in the testis can cause
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apoptosis of spermatogenic cells to further worsen male reproductive function [15,
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16]. For example environmental pollutants including such as cadmium decreased
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sperm motility and counts, caused morphologic abnormalities and promoted
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expression of Bax and Bcl-2 as well as caspase-3 in males [15, 17].
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The animal models are commonly used for the study of human infertility. The
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methods of creating sterility models include physical, chemical and endocrine factors.
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Chemical inducers include gossypol [18], adenine induction [19], formaldehyde [20]
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and busulfan [21]. The advantage of the busulfan-induced sterile mouse model is that
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the dysfunction of reproductive system and infertility is similar in humans and
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animals [22]. Busulfan has been successfully used in the generation of a mouse
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sterility model [23]. Excessive busulfan intake interferes with hormone synthesis and
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metabolism related to normal reproductive function, fetal development and testicular
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atrophy, thereby resulting in boty male and female reproductive dysfunction [24, 25].
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Molybdenum (Mo) plays an important role in maintaining normal metabolism in
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humans and animals; it is one of the essential trace elements of the body [26, 27]. It
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catalyzes hydroxylation, oxygen atom transfer and other oxidation-reduction reactions
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to promote the metabolism of of substrates and discharge of some metabolites [28,
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29]. Our previous studies showed that Mo has both nutritional and toxic effects on
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mouse sperm and oocytes, as well as on testicular and ovarian oxidative stress in a 3
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dose-dependent manner. At a low dose, Mo improved sperm and oocyte quality, but at
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a high dose it damaged testicular and ovarian function. The dose of 25 mg/L Mo
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significantly increased the reproductive parameters in male mice [30]. Therefore, we
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hypothesized that Mo may play a therapeutic role in the male infertility.
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In the present study, a male sterile mouse model was generated by intraperitoneal
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injection of busulfan. Changes in reproductive hormone levels, testicular structures,
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oxidative stress and apoptosis in the experimental and control groups were measured,
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and the therapeutic effects of Mo on male sterility were explored, so as to identify the
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treatments for male sterility for the benefit of human reproductive health.
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2. Materials and methods
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This animal experiment was approved by the Institutional Animal Care and Use
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Committee (IACUC) of Henan University of Science and Technology. All animals
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were humanely handled. 2.1 Chemicals
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Unless otherwise stated, all components used in the present study were procured
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from Sigma-Aldrich Corp. (St. Louis, MO, USA).
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2.2 Animals
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Healthy male Kunming mice (42-day-old), weighing at 24±0.5 g were obtained
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from the Experimental Animal Center of Zhengzhou University, and were maintained
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in a standard animal house with air conditioning. Then, 90 mice were randomly
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divided into control, busulfan and busulfan+Mo groups of 30 each. Mice in each 4
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group were given a standard diet during the treatment period.
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2.3 Exposure of mice to busulfan
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Mice in the control group received a single intraperitoneal injection of 50%
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DMSO. The busulfan and busulfan+Mo groups received a single intraperitoneal
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injection of busulfan (20 mg/kg body weight) diluted in DMSO.
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2.4 Treatment of mice to molybdenum
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After 30 days, 30 mice in busulfan+Mo group were given drinking water
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containing 20 mg/L Mo for continuously 42 days. The remaining mice drank normal
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water. Thirty mice (10 per group) were sacrificed and used for subsequent
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experiments. The remaining 60 mice were mated with female mice in spontaneous
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estrus at 72 Day after treatment.
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2.5 Collection of testes and epididymides
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The testes of mice from each group were used for determination of SOD, LDH
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and MDA. The mice were sacrificed by cervical dislocation. Testes and epididymides
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were quickly removed and weighed. Testes was placed in 4 °C precooled saline in a
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refrigerator, and then transferred to -20 °C before homogenate preparation.
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2.6 Evaluation of coefficient of testis and epididymis
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The weights of testes and epididymides in each group were measured, and then
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the organ coefficient was calculated according to the following equation: coefficient =
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wet weight of organ (mg)/body weight (g)×100%.
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2.7 Histopathological observation
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HE staining was performed to observe the morphological structure changes of
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the testes. Testis tissues from three groups were removed and quickly fixed in 10%
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formaldehyde, immersed in 10% neutral formalin for 24 h, rinsed with distilled water,
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dehydrated in graded alcohol (75%, 85%, 95% and 100%), then cleared in xylene, and
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embedded in paraffin. Blocks were cut into 5-μm sections with a rotary microtome
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and sections were stained with hematoxylin and eosin.
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2.8 Detection the levels of FSH, LH, T and E2 in serum
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Blood samples were gained from the eyeballs after absolute diet 12 hours.
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ELISA kits (Haling Biotechnology Co. Ltd., Shanghai, China) were used to measure
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the concentrations of FSH, LH, T and E2 in testis tissues according to the
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manufacturer’s recommendations.
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2.9 Measurement of SOD, LDH and MDA levels in testes
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Three biochemical markers (SOD, LDH and MDA) were spectrophotometrically
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measured following the instructions of commercially available diagnostic kits
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(Nanjing Jiancheng Bioengineering Institute of China). Testis tissues were
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homogenized in cold PBS using an automatic homogenizer and were centrifuged at
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3000g for 5 min. Supernatants were collected to measure SOD, LDH and MDA
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levels.
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2.10 Immunohistochemical staining analysis Immunohistochemical staining was carried out and modified according to
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methods reported by Chen [31]. Briefly, paraffin-embedded testis tissues were serially
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sectioned at 5 μm. They were dewaxed rehydrated in xylene and distilled water, and
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heated in a microwave oven. Endogenous peroxidase activity was quenched with 3%
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hydrogen peroxide for 10 min, and sections were blocked with 5% (v/v) bovine
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normal serum in phosphate-buffered saline (PBS) for 25 min. Thereafter, the sections
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were incubated overnight at 4 °C in a humidified atmosphere with specific primary
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antibodies against Bax, Bcl-2, caspase-3 and caspase-9. Subsequently, the sections
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were washed in PBS three times and were incubated with secondary antibody.
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Staining of the sections was observed by adding 3,3'-diaminobenzidine (DAB). The
136
sections were counterstained with hematoxylin and rinsed in tap water.
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Immunohistochemical micrographs were taken with a microscope camera system.
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2.11 Western blot analysis
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The western blot technique was used to analyze the expression of Bax, Bcl-2
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caspase-3 and caspase-9 protein. Fresh testis tissues were homogenized, centrifuged
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and supernatant proteins were collected. The samples were subjected to SDS-PAGE
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and were then transferred to PVDF membranes. Membranes were blocked in 5% non-
143
fat milk, and were incubated with primary antibodies against Bax, Bcl-2, caspase-3
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and caspase-9 and then secondary antibodies. Positive bands were measured using
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enhanced chemiluminescence reagents.β-actin density was used as a control to
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normalized gray densities of the protein bands.
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2.12 Recording number of births and gender distribution After treatment for 72 days, the male mice in each group were mated with female
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mice in spontaneous estrus. After pregnancy and delivery, the number of births and
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gender distributions were recorded.
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2.13 Statistical analysis
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Five replicates in each experiment group were conducted. All data were tested
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using a single variable of the general linear model using SPSS Statistics 17.
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Differences among experimental groups were considered significant at P < 0.05 and
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very significant at P < 0.01.
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3 Results
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Effects of Mo on the coefficients of testis and epididymis, hormones levels,
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oxidative stress and numbers of reproductive parameters in each group male mice are
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shown in Table 1. The coefficients of testis and epididymis in busulfan+Mo group
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were consistent with those of the control group but were significantly higher than the
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those of busulfan group (P < 0.01). Levels of T (P < 0.01) and E2 (P < 0.05) in the
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busulfan group were significantly lower, and LH was significantly higher (P < 0.01)
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than those of the control group. Levels of T and E2 in busulfan+Mo group were
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significantly higher and LH levels was significantly lower (P < 0.01) than those of the
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busulfan group. Levels of E2 and LH were not significantly different from those of the
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control group. T levels were significantly higher (P < 0.01) than those of the control
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group. Levels of FSH showed no apparent differences among the three groups. The
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busulfan group had higher levels of LDH (P < 0.01) and MDA (P < 0.05) and lower
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levels of SOD (P < 0.01) than did the control group. The busulfan+Mo group had
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significantly higher SOD levels (P < 0.01) and lower LDH (P < 0.01) and MDA 8
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levels (P < 0.05) than did the busulfan group. The busulfan+Mo group had similar
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levels of SOD and MDA as did the control group. This group also had higher LDH
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levels (P < 0.05) than those of the control group. In the busulfan group, pregnancy
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rates (P < 0.01), No. offspring (P < 0.01) and average No. offspring (P < 0.01) were
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significantly lower than those of the control group. In the busulfan+Mo group, the
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pregnancy rate (P < 0.01), No. offspring (P < 0.01) and average No. offspring (P <
177
0.01) were significantly higher than those of the busulfan group. All the indicators in
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the busulfan+Mo group were not significantly different from those of the control
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group.
180
Effects of Mo on the histology structure of testis in each group male mice are
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shown in Fig 1. Testicular morphology in the busulfan+Mo group was similar to that
182
of the control group, showing regular arrangement of seminiferous tubules and
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spermatogenic cells. The busulfan group showed thinner seminiferous tubules, with
184
missing tubules and germ cells in the basement membrane.
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Effects of Mo on the expression of Bax, Bcl-2, caspase-3 and caspase-9 in each
186
group male mice are shown in Fig 2. The expression of Bax, Bcl-2, caspase-3 and
187
caspase-9 were weakly positive in the control and busulfan+Mo groups, but were
188
strongly positive in the busulfan group. The differences between the control and
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busulfan+Mo groups and the busulfan group were remarkable. Western blotting (Fig
190
3) showed similar protein expression patterns.
191
4. Discussion A sterile mile mouse model was successfully generated by a single
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intraperitoneal injection of busulfan, consistent with the results of our previous study
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[32]. Thirty days after injection, the mice were mated with female mice in
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spontaneous estrus, but they did not become pregnant, suggesting that the sterile male
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mouse model was successfully induced by busulfan.
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The mechanism of busulfan-induced mice sterility may be related to changes
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intesticular structure, reproductive hormone levels, oxidative stress, or apoptosis.
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After exposure to busulfan, the mice demonstrated thinner seminiferous tubules,
200
vanishing tubules and germ cells in the basement membrane, decreased coefficients of
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the testis and epididymis, decreased levels of T, E2 and SOD, increased levels of LH,
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LDH and MDA, and increased expression of Bax, Bcl-2, caspase-3 and caspase-9.
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Other studies reported that busulfan increased number of apoptotic sperm and levels
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of LDH, leading to SOD depletion and induction of oxidative stress, decreasing the
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serum concentrations of T [33, 34]. These results are consistent with those of the
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present study. Busulfan decreased Bcl-2 expression and increased Bax expression in
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sperm; howerer, in our study, expression levels of Bcl-2 were increased by busulfan.
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This was probably a consequence of inhibiting apoptosis.
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In the present study, Mo recovered the reproductive function of sterile mice in
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terms of macroscopic pregnancy rate and offspring number, as well as microscopic
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reproductive parameters. After the addition of Mo to sterile mice for 42 days, the
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coefficients of the testes and epididymides, histomorphological structure of the testes
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and the levels of T, E2 and LH recovered to almost normal levels. This may have been
214
associated with molybdenum promoting the proliferation of supporting cells and 10
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interstitial cells in the testis [30]. Studies indicated that appropriate amount of Mo can
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promote the synthesis of DNA and further accelerate cell proliferation [29]. Zhai et al.
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reported that Mo at 25 mg/L increased the sperm parameters, including the
218
epididymis index, sperm motility, sperm count, and morphology [30], in agreement
219
with results in the present study. Unfortunately, we could not find any reference
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regarding Mo improving the reproductive hormone levels in male mice to date;
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therefore, we could not compare the levels of T, E2, LH and FSH to those of other
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studies. Nevertheless, in the present study, Mo restored the levels of T, E2 and LH to
223
the almost normal.
224
Molybdenum (Mo) rescued sterile mice from oxidative damage induced by
225
busulfan to normal levels, as shown by levels of SOD, LDH and MDA. Oxidative
226
stress produces
227
resulting in damage to nucleic acid, protein, carbohydrate and lipids composition of
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cell damage [35, 36]. The resulting disturbances in energy metabolism, tricarboxylic
229
acid cycle, oxidative phosphorylation and glycolysis decrease spermatogenic function
230
[37, 38]. This agrees our results regarding SOD, MDA and LDH levels. The
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restorative effects of Mo on reproductive function could be due to promotion of
232
metabolism of substances and the discharge of other metabolites by catalyzing
233
hydroxylation, oxygen atom transfer and other oxidation-reduction reactions [39].
234
These data suggest that moderate amounts of molybdenum could be helpful to resist
235
oxidative stress.
strong cytotoxic effects in testis in the form of lipid peroxidation,
Oxidative stress is accompanied by apoptosis. Apoptosis involves a series of
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gene activations, gene expressions and regulations [40, 41]. In the present study, we
238
found that abnormal expression levels of Bax, Bcl-2, caspase-3 and caspase-9 in the
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testis of sterile mice were significantly restored to normal levels by adding Mo into
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drinking water. The possible mechanism could be that molybdenum interfered with
241
the expression of cytochrome c by promoting the production of cytochrome c
242
reductase [42]. Studies have indicated that cytochrome c combined with Apaf-1 may
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form apoptotic bodies, resulting in the activation of caspase-3 and caspase-9 [43, 44].
244
As cytochrome c is upstream of caspase-3 and caspase-9, decreases in cytochrome c
245
levels may mediate inhibition of transcription and expression of downstream primers
246
[45]. The caspase cascade leads to fragmentation of cell DNA [46, 47]. In contrast,
247
the decrease in caspase family members mediates inhibition of apoptosis by
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increasing the permeability of the cell membrane and disturbing the formation of
249
DNA. Furthermore, Bax combines with Bcl-2 to form a heterogeneous two polymer
250
structure that blocks the function of inhibiting apoptosis of Bcl-2 [48-50]. Therefore,
251
our data suggested that Mo may interfere with the expression of pro-apoptotic factors.
252
Finally, after Mo treatment for 42 days, the sterile mice impregnated females
253
who delivered offspring with normal number and gender distribution. To the best of
254
our knowledge, this was the first report on the effects of Mo treatment of sterile mice
255
up until birth of offspring.
256
In conclusion, Mo at 20 mg/L has a significant recovery restorative effect on
257
injured testes, and has substantial therapeutic effects on reproductive dysfunction in
258
sterile mice. The mechanism of Mo therapeutic effect on sterile mice may related to 12
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repair of damaged testicular structure, normalization of regulate abnormal
260
reproductive hormone levels, decreased oxidative stress levels or resistance to
261
apoptosis. Therefore, Mo may be a new candidate treatment for infertility.
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Conflict of Interest The authors declare no conflict of interest.
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Acknowledgements This work is sponsored by the National Natural Science Foundation of China
265 266
(31402153).
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433 434
Fig. 1. Effects of busulfan or Mo on the histology structure of testis in sterile model mice. (n=30)
435
A1 and A2: control group; B1 and B2: busulfan group; C1 and C2: busulfan +Mo group.
436
A1 and A2 presented tubules with thin basement membrane and tunica propria as well as normal
437
germinal epithelium showing orderly progression from spermatogonia to spermatocytes with
438
groups of spermatids and mature spermatozoa;
439
B1 and B2 indicated that there were a substantial number of vacuoles located in the seminiferous
440
tubules, spermatogenic cells were completely lost in busulfan group;
441
C1 and C2: Spermatocytes exhibited partial survival in testis that were administered Mo.
ACCEPTED MANUSCRIPT
442 443
Fig. 2. Immunohistochemical staining image on the expression of Bax, Bcl-2, caspase-3 and
444
caspase-9 in the testis. (n=30)
445
A, B, C and D represented the expression of Bax, Bcl-2, caspase-3 and caspase-9 in turn;
446
1, 2 and 3 represented the control group, the busulfan group, the busulfan+Mo group in turn;
447
For example: A1 represented the expression of Bax in the control group. Immunohistochemistry
448
demonstrated that Bax, Bcl-2, caspase-3 and caspase-9 protein were not almost expressed in testis
449
(A1, B1, C1 and D1); Bax, Bcl-2, caspase-3 and caspase-9 protein were widely expressed in
450
spermatogenic cells (A2, B2, C2 and D2); Bax, Bcl-2, caspase-3 and caspase-9 protein were
451
weakly expressed in spermatogenic cells (A3, B3, C3 and D3).
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452 453
Fig. 3. Mo relieved busulfan-induced apoptosis on the expression of Bax, Bcl-2, caspase-3 and
454
caspase-9 in the testis. (n=30)
455
A1: Western blot analysis the expression of Bax, Bcl-2, caspase-3 and caspase-9; A2: Image-J
456
software calculated the gray value of Bax, Bcl-2, caspase-3 and caspase-9.
457
column, values without a common capital superscript differ (P < 0.01). a,b Within a column, values
458
without a common capital superscript differ (P < 0.05). Bus: busulfan Group; Bus+Mo:
459
busulfan+Mo group.
A,B,C
Within each
ACCEPTED MANUSCRIPT In this work, our results indicated Mo at 20 mg/L has a significant recovery effect on the injured testis, and it has obvious therapeutic effect on reproductive dysfunction of infertility mice. The mechanism of Mo therapeutic effect on mice infertility could be due to that Mo could repair the damaged testicular structure, regulate abnormal reproductive endocrine, decrease oxidative stress, and resist cell apoptosis. To our knowledge, this is the first report on the offspring birth to term of Mo treating infertility mice.
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Table. 1 Effects of Mo on the organ coefficient, hormones levels, oxidative stress and reproductive parameters in each group male mice. (n=30) Control Organ coefficient
Hormones levels
Oxidative stress
Reproductive parameters
Bus+Mo
Testis
5.501±0.444A
2.643±0.682B
5.563±1.084A
Epididymis
2.478±0.085A
2.259±0.096B
2.459±0.571A
T (ng/L)
12.66±0.90A
10.56±0.28B
13.52±1.29C
FSH (IU/L)
1.07±0.08
1.16±0.11
1.02±0.04
LH (ng/L)
194.89±6.16A
205.18±3.90B
190.40±3.90A
E2(ng/L)
2.82±0.69Aa
2.10±0.10B
2.52±0.11Ab
SOD(U/mgprot)
249.22±22.42A
211.60±14.71B
251.22±31.46A
LDH(U/gprot)
2708.86±123.53Aa
4616.88±129.25B
2818.293±193.21Ab
MDA(nmol/mgprot)
1.02±0.12a
1.19±0.16b
1.03±0.18c
Pregnancy rate
1A (20/20)
0.05B (1/20)
0.95A(19/20)
No. offspring
215A
6B
208A
1:0.97
1:1
1:1.02
10.75A
6B
10.95A
Offspring gender ratio(Male: Female) Average No. offspring 424 425 426 427 428 429 430 431 432
Bus
Data are reported as means (±SD). A,B,C Within a line, values without a common capital superscript differ (P < 0.01) ; a,b,c Within a line, values without a common lowercase superscript differ (P < 0.05). Bus: busulfan group; Bus+Mo: busulfan+Mo group. Control: mice received a single intraperitoneal injection of 50% DMSO at 42 days of age. Bus: mice received a single intraperitoneal injection of busulfan (20 mg/kg body weight) diluted in DMSO at 42 days of age. Bus+Mo: mice received a single intraperitoneal injection of busulfan (20 mg/kg body weight) diluted in DMSO at 42 days of age and were given drinking water containing 20 mg/L Mo at 72 days of age for continuously 42 days. Organ coefficient= wet organ weight/totle wet body weight (mg/g). SOD: superoxide dismutase, LDH: lactate dehydrogenase, MDA: malondialdehyde.
Feng-Jun Liu, Wen-Ya Dong, Hao Zhao, Xin-Huai Shi, Yu-Ling Zhang PII:
S0093-691X(18)31087-2
DOI:
10.1016/j.theriogenology.2018.12.002
Reference:
THE 14794
To appear in:
Theriogenology
Received Date:
17 April 2018
Accepted Date:
01 December 2018
Please cite this article as: Feng-Jun Liu, Wen-Ya Dong, Hao Zhao, Xin-Huai Shi, Yu-Ling Zhang, Effect of molybdenum on reproductive function of male mice treated with busulfan, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.12.002
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ACCEPTED MANUSCRIPT 1 2
Effect of molybdenum on reproductive function of male mice treated with busulfan
3
Feng-Jun Liu*, Wen-Ya Dong, Hao Zhao, Xin-Huai Shi, Yu-Ling Zhang
4
Animal College of Science and Technology, Henan University of Science and
5
Technology, Luoyang 471003, PR China.
6
Abstract
7
Infertility is a serious public health problem worldwide. Molybdenum (Mo)
8
plays an important role in maintaining normal metabolism. To explore the therapeutic
9
efficacy of molybdenum (Mo) on male infertility, 90 mice were randomly divided
10
into control, busulfan and busulfan+Mo groups. The male mice in the busulfan and
11
busulfan+Mo groups were exposed to busulfan (20 mg/kg body weight) with a single
12
intraperitoneal injection to establish the infertility model. The sterile mice were
13
successfully obtained 30 days after busulfan exposure. Then, the male mice in the
14
busulfan+Mo group were given drinking water containing 20 mg/L Mo continuously
15
for 42 days. At 72 Day after treatment, 30 mice in the three groups were tested for
16
various indices, and 60 mice were mated with females in spontaneous estrus. Mo
17
significantly reversed the thinner seminiferous tubules and disappeared tubule and
18
germ cells. Mo also normalized previously abnormal levels of testosterone, estradiol,
19
luteinizing hormone, superoxide dismutase, lactate dehydrogenase, malondialdehyde.
20
Furthermore, expression levels expression of Bax, Bcl-2, caspase-3 and caspase-9 * Corresponding author at: College of Animal Science and Technology, Henan University of Science and Technology, No.263 Kaiyuan Avenue, Luoyang, China, 471023. Email: [email protected] (Feng-jun Liu) 1
Revised
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returned to control levels; and finally, Mo-treated sterile mice obtained offspring with
22
normal number and gender ratio. These results suggested that Mo at 20 mg/L had a
23
significant therapeutic effect on reproductive dysfunction in sterile mice. Its
24
mechanism could via repair of damaged testicular structures, regulation of abnormal
25
reproductive hormone levels, decreased oxidative stress or and resistance to cell
26
apoptosis. Mo may be a new candidate medicine for treatment of male infertility.
27
Key- words: molybdenum; busulfan; infertility; testis; mouse
28
1. Introduction
29
Infertility is a serious health problem worldwide, and its incidence is increasing.
30
Currently, the incidence of the infertility is approximately 24% in humans, with 18%
31
in the men [1]. Infertility may be caused by a variety of factors, including disease,
32
environmental pollution, endocrine disorders and reproductive system injuries [2-5].
33
The testis is the only organ that generates sperm in the male reproductive system. Its
34
normal structure and function are important for the maintenance of male reproduction
35
[6]. Therefore, any disturbance to the testis may result in male infertility.
36
Reproductive hormone alterations, apoptosis, oxidative damage and abnormal tissue
37
structures in the testis and epididymis might be mechanisms for the degeneration or
38
disappearance of male reproductive function [7-9]. The abnormal synthesis and
39
metabolism of reproductive hormones, such as FSH (follicle stimulating hormone),
40
luteinizing hormone (LH), testosterone (T) and estradiol (E2) could cause
41
reproductive disorders [10-12]. Imbalances in redox states involving superoxide 2
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dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA)
43
have been shown to mediate male infertility associated with many diseases [13, 14].
44
Furthermore, the high expression of apoptotic factors in the testis can cause
45
apoptosis of spermatogenic cells to further worsen male reproductive function [15,
46
16]. For example environmental pollutants including such as cadmium decreased
47
sperm motility and counts, caused morphologic abnormalities and promoted
48
expression of Bax and Bcl-2 as well as caspase-3 in males [15, 17].
49
The animal models are commonly used for the study of human infertility. The
50
methods of creating sterility models include physical, chemical and endocrine factors.
51
Chemical inducers include gossypol [18], adenine induction [19], formaldehyde [20]
52
and busulfan [21]. The advantage of the busulfan-induced sterile mouse model is that
53
the dysfunction of reproductive system and infertility is similar in humans and
54
animals [22]. Busulfan has been successfully used in the generation of a mouse
55
sterility model [23]. Excessive busulfan intake interferes with hormone synthesis and
56
metabolism related to normal reproductive function, fetal development and testicular
57
atrophy, thereby resulting in boty male and female reproductive dysfunction [24, 25].
58
Molybdenum (Mo) plays an important role in maintaining normal metabolism in
59
humans and animals; it is one of the essential trace elements of the body [26, 27]. It
60
catalyzes hydroxylation, oxygen atom transfer and other oxidation-reduction reactions
61
to promote the metabolism of of substrates and discharge of some metabolites [28,
62
29]. Our previous studies showed that Mo has both nutritional and toxic effects on
63
mouse sperm and oocytes, as well as on testicular and ovarian oxidative stress in a 3
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dose-dependent manner. At a low dose, Mo improved sperm and oocyte quality, but at
65
a high dose it damaged testicular and ovarian function. The dose of 25 mg/L Mo
66
significantly increased the reproductive parameters in male mice [30]. Therefore, we
67
hypothesized that Mo may play a therapeutic role in the male infertility.
68
In the present study, a male sterile mouse model was generated by intraperitoneal
69
injection of busulfan. Changes in reproductive hormone levels, testicular structures,
70
oxidative stress and apoptosis in the experimental and control groups were measured,
71
and the therapeutic effects of Mo on male sterility were explored, so as to identify the
72
treatments for male sterility for the benefit of human reproductive health.
73
2. Materials and methods
74
This animal experiment was approved by the Institutional Animal Care and Use
75
Committee (IACUC) of Henan University of Science and Technology. All animals
76
were humanely handled. 2.1 Chemicals
77
Unless otherwise stated, all components used in the present study were procured
78 79
from Sigma-Aldrich Corp. (St. Louis, MO, USA).
80
2.2 Animals
81
Healthy male Kunming mice (42-day-old), weighing at 24±0.5 g were obtained
82
from the Experimental Animal Center of Zhengzhou University, and were maintained
83
in a standard animal house with air conditioning. Then, 90 mice were randomly
84
divided into control, busulfan and busulfan+Mo groups of 30 each. Mice in each 4
ACCEPTED MANUSCRIPT 85
group were given a standard diet during the treatment period.
86
2.3 Exposure of mice to busulfan
87
Mice in the control group received a single intraperitoneal injection of 50%
88
DMSO. The busulfan and busulfan+Mo groups received a single intraperitoneal
89
injection of busulfan (20 mg/kg body weight) diluted in DMSO.
90
2.4 Treatment of mice to molybdenum
91
After 30 days, 30 mice in busulfan+Mo group were given drinking water
92
containing 20 mg/L Mo for continuously 42 days. The remaining mice drank normal
93
water. Thirty mice (10 per group) were sacrificed and used for subsequent
94
experiments. The remaining 60 mice were mated with female mice in spontaneous
95
estrus at 72 Day after treatment.
96
2.5 Collection of testes and epididymides
97
The testes of mice from each group were used for determination of SOD, LDH
98
and MDA. The mice were sacrificed by cervical dislocation. Testes and epididymides
99
were quickly removed and weighed. Testes was placed in 4 °C precooled saline in a
100
refrigerator, and then transferred to -20 °C before homogenate preparation.
101
2.6 Evaluation of coefficient of testis and epididymis
102
The weights of testes and epididymides in each group were measured, and then
103
the organ coefficient was calculated according to the following equation: coefficient =
104
wet weight of organ (mg)/body weight (g)×100%.
105 5
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106
2.7 Histopathological observation
107
HE staining was performed to observe the morphological structure changes of
108
the testes. Testis tissues from three groups were removed and quickly fixed in 10%
109
formaldehyde, immersed in 10% neutral formalin for 24 h, rinsed with distilled water,
110
dehydrated in graded alcohol (75%, 85%, 95% and 100%), then cleared in xylene, and
111
embedded in paraffin. Blocks were cut into 5-μm sections with a rotary microtome
112
and sections were stained with hematoxylin and eosin.
113
2.8 Detection the levels of FSH, LH, T and E2 in serum
114
Blood samples were gained from the eyeballs after absolute diet 12 hours.
115
ELISA kits (Haling Biotechnology Co. Ltd., Shanghai, China) were used to measure
116
the concentrations of FSH, LH, T and E2 in testis tissues according to the
117
manufacturer’s recommendations.
118
2.9 Measurement of SOD, LDH and MDA levels in testes
119
Three biochemical markers (SOD, LDH and MDA) were spectrophotometrically
120
measured following the instructions of commercially available diagnostic kits
121
(Nanjing Jiancheng Bioengineering Institute of China). Testis tissues were
122
homogenized in cold PBS using an automatic homogenizer and were centrifuged at
123
3000g for 5 min. Supernatants were collected to measure SOD, LDH and MDA
124
levels.
125
2.10 Immunohistochemical staining analysis Immunohistochemical staining was carried out and modified according to
126 6
ACCEPTED MANUSCRIPT 127
methods reported by Chen [31]. Briefly, paraffin-embedded testis tissues were serially
128
sectioned at 5 μm. They were dewaxed rehydrated in xylene and distilled water, and
129
heated in a microwave oven. Endogenous peroxidase activity was quenched with 3%
130
hydrogen peroxide for 10 min, and sections were blocked with 5% (v/v) bovine
131
normal serum in phosphate-buffered saline (PBS) for 25 min. Thereafter, the sections
132
were incubated overnight at 4 °C in a humidified atmosphere with specific primary
133
antibodies against Bax, Bcl-2, caspase-3 and caspase-9. Subsequently, the sections
134
were washed in PBS three times and were incubated with secondary antibody.
135
Staining of the sections was observed by adding 3,3'-diaminobenzidine (DAB). The
136
sections were counterstained with hematoxylin and rinsed in tap water.
137
Immunohistochemical micrographs were taken with a microscope camera system.
138
2.11 Western blot analysis
139
The western blot technique was used to analyze the expression of Bax, Bcl-2
140
caspase-3 and caspase-9 protein. Fresh testis tissues were homogenized, centrifuged
141
and supernatant proteins were collected. The samples were subjected to SDS-PAGE
142
and were then transferred to PVDF membranes. Membranes were blocked in 5% non-
143
fat milk, and were incubated with primary antibodies against Bax, Bcl-2, caspase-3
144
and caspase-9 and then secondary antibodies. Positive bands were measured using
145
enhanced chemiluminescence reagents.β-actin density was used as a control to
146
normalized gray densities of the protein bands.
147
2.12 Recording number of births and gender distribution After treatment for 72 days, the male mice in each group were mated with female
148 7
ACCEPTED MANUSCRIPT 149
mice in spontaneous estrus. After pregnancy and delivery, the number of births and
150
gender distributions were recorded.
151
2.13 Statistical analysis
152
Five replicates in each experiment group were conducted. All data were tested
153
using a single variable of the general linear model using SPSS Statistics 17.
154
Differences among experimental groups were considered significant at P < 0.05 and
155
very significant at P < 0.01.
156
3 Results
157
Effects of Mo on the coefficients of testis and epididymis, hormones levels,
158
oxidative stress and numbers of reproductive parameters in each group male mice are
159
shown in Table 1. The coefficients of testis and epididymis in busulfan+Mo group
160
were consistent with those of the control group but were significantly higher than the
161
those of busulfan group (P < 0.01). Levels of T (P < 0.01) and E2 (P < 0.05) in the
162
busulfan group were significantly lower, and LH was significantly higher (P < 0.01)
163
than those of the control group. Levels of T and E2 in busulfan+Mo group were
164
significantly higher and LH levels was significantly lower (P < 0.01) than those of the
165
busulfan group. Levels of E2 and LH were not significantly different from those of the
166
control group. T levels were significantly higher (P < 0.01) than those of the control
167
group. Levels of FSH showed no apparent differences among the three groups. The
168
busulfan group had higher levels of LDH (P < 0.01) and MDA (P < 0.05) and lower
169
levels of SOD (P < 0.01) than did the control group. The busulfan+Mo group had
170
significantly higher SOD levels (P < 0.01) and lower LDH (P < 0.01) and MDA 8
ACCEPTED MANUSCRIPT 171
levels (P < 0.05) than did the busulfan group. The busulfan+Mo group had similar
172
levels of SOD and MDA as did the control group. This group also had higher LDH
173
levels (P < 0.05) than those of the control group. In the busulfan group, pregnancy
174
rates (P < 0.01), No. offspring (P < 0.01) and average No. offspring (P < 0.01) were
175
significantly lower than those of the control group. In the busulfan+Mo group, the
176
pregnancy rate (P < 0.01), No. offspring (P < 0.01) and average No. offspring (P <
177
0.01) were significantly higher than those of the busulfan group. All the indicators in
178
the busulfan+Mo group were not significantly different from those of the control
179
group.
180
Effects of Mo on the histology structure of testis in each group male mice are
181
shown in Fig 1. Testicular morphology in the busulfan+Mo group was similar to that
182
of the control group, showing regular arrangement of seminiferous tubules and
183
spermatogenic cells. The busulfan group showed thinner seminiferous tubules, with
184
missing tubules and germ cells in the basement membrane.
185
Effects of Mo on the expression of Bax, Bcl-2, caspase-3 and caspase-9 in each
186
group male mice are shown in Fig 2. The expression of Bax, Bcl-2, caspase-3 and
187
caspase-9 were weakly positive in the control and busulfan+Mo groups, but were
188
strongly positive in the busulfan group. The differences between the control and
189
busulfan+Mo groups and the busulfan group were remarkable. Western blotting (Fig
190
3) showed similar protein expression patterns.
191
4. Discussion A sterile mile mouse model was successfully generated by a single
192 9
ACCEPTED MANUSCRIPT 193
intraperitoneal injection of busulfan, consistent with the results of our previous study
194
[32]. Thirty days after injection, the mice were mated with female mice in
195
spontaneous estrus, but they did not become pregnant, suggesting that the sterile male
196
mouse model was successfully induced by busulfan.
197
The mechanism of busulfan-induced mice sterility may be related to changes
198
intesticular structure, reproductive hormone levels, oxidative stress, or apoptosis.
199
After exposure to busulfan, the mice demonstrated thinner seminiferous tubules,
200
vanishing tubules and germ cells in the basement membrane, decreased coefficients of
201
the testis and epididymis, decreased levels of T, E2 and SOD, increased levels of LH,
202
LDH and MDA, and increased expression of Bax, Bcl-2, caspase-3 and caspase-9.
203
Other studies reported that busulfan increased number of apoptotic sperm and levels
204
of LDH, leading to SOD depletion and induction of oxidative stress, decreasing the
205
serum concentrations of T [33, 34]. These results are consistent with those of the
206
present study. Busulfan decreased Bcl-2 expression and increased Bax expression in
207
sperm; howerer, in our study, expression levels of Bcl-2 were increased by busulfan.
208
This was probably a consequence of inhibiting apoptosis.
209
In the present study, Mo recovered the reproductive function of sterile mice in
210
terms of macroscopic pregnancy rate and offspring number, as well as microscopic
211
reproductive parameters. After the addition of Mo to sterile mice for 42 days, the
212
coefficients of the testes and epididymides, histomorphological structure of the testes
213
and the levels of T, E2 and LH recovered to almost normal levels. This may have been
214
associated with molybdenum promoting the proliferation of supporting cells and 10
ACCEPTED MANUSCRIPT 215
interstitial cells in the testis [30]. Studies indicated that appropriate amount of Mo can
216
promote the synthesis of DNA and further accelerate cell proliferation [29]. Zhai et al.
217
reported that Mo at 25 mg/L increased the sperm parameters, including the
218
epididymis index, sperm motility, sperm count, and morphology [30], in agreement
219
with results in the present study. Unfortunately, we could not find any reference
220
regarding Mo improving the reproductive hormone levels in male mice to date;
221
therefore, we could not compare the levels of T, E2, LH and FSH to those of other
222
studies. Nevertheless, in the present study, Mo restored the levels of T, E2 and LH to
223
the almost normal.
224
Molybdenum (Mo) rescued sterile mice from oxidative damage induced by
225
busulfan to normal levels, as shown by levels of SOD, LDH and MDA. Oxidative
226
stress produces
227
resulting in damage to nucleic acid, protein, carbohydrate and lipids composition of
228
cell damage [35, 36]. The resulting disturbances in energy metabolism, tricarboxylic
229
acid cycle, oxidative phosphorylation and glycolysis decrease spermatogenic function
230
[37, 38]. This agrees our results regarding SOD, MDA and LDH levels. The
231
restorative effects of Mo on reproductive function could be due to promotion of
232
metabolism of substances and the discharge of other metabolites by catalyzing
233
hydroxylation, oxygen atom transfer and other oxidation-reduction reactions [39].
234
These data suggest that moderate amounts of molybdenum could be helpful to resist
235
oxidative stress.
strong cytotoxic effects in testis in the form of lipid peroxidation,
Oxidative stress is accompanied by apoptosis. Apoptosis involves a series of
236 11
ACCEPTED MANUSCRIPT 237
gene activations, gene expressions and regulations [40, 41]. In the present study, we
238
found that abnormal expression levels of Bax, Bcl-2, caspase-3 and caspase-9 in the
239
testis of sterile mice were significantly restored to normal levels by adding Mo into
240
drinking water. The possible mechanism could be that molybdenum interfered with
241
the expression of cytochrome c by promoting the production of cytochrome c
242
reductase [42]. Studies have indicated that cytochrome c combined with Apaf-1 may
243
form apoptotic bodies, resulting in the activation of caspase-3 and caspase-9 [43, 44].
244
As cytochrome c is upstream of caspase-3 and caspase-9, decreases in cytochrome c
245
levels may mediate inhibition of transcription and expression of downstream primers
246
[45]. The caspase cascade leads to fragmentation of cell DNA [46, 47]. In contrast,
247
the decrease in caspase family members mediates inhibition of apoptosis by
248
increasing the permeability of the cell membrane and disturbing the formation of
249
DNA. Furthermore, Bax combines with Bcl-2 to form a heterogeneous two polymer
250
structure that blocks the function of inhibiting apoptosis of Bcl-2 [48-50]. Therefore,
251
our data suggested that Mo may interfere with the expression of pro-apoptotic factors.
252
Finally, after Mo treatment for 42 days, the sterile mice impregnated females
253
who delivered offspring with normal number and gender distribution. To the best of
254
our knowledge, this was the first report on the effects of Mo treatment of sterile mice
255
up until birth of offspring.
256
In conclusion, Mo at 20 mg/L has a significant recovery restorative effect on
257
injured testes, and has substantial therapeutic effects on reproductive dysfunction in
258
sterile mice. The mechanism of Mo therapeutic effect on sterile mice may related to 12
ACCEPTED MANUSCRIPT 259
repair of damaged testicular structure, normalization of regulate abnormal
260
reproductive hormone levels, decreased oxidative stress levels or resistance to
261
apoptosis. Therefore, Mo may be a new candidate treatment for infertility.
262
Conflict of Interest The authors declare no conflict of interest.
263 264
Acknowledgements This work is sponsored by the National Natural Science Foundation of China
265 266
(31402153).
267
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433 434
Fig. 1. Effects of busulfan or Mo on the histology structure of testis in sterile model mice. (n=30)
435
A1 and A2: control group; B1 and B2: busulfan group; C1 and C2: busulfan +Mo group.
436
A1 and A2 presented tubules with thin basement membrane and tunica propria as well as normal
437
germinal epithelium showing orderly progression from spermatogonia to spermatocytes with
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groups of spermatids and mature spermatozoa;
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B1 and B2 indicated that there were a substantial number of vacuoles located in the seminiferous
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tubules, spermatogenic cells were completely lost in busulfan group;
441
C1 and C2: Spermatocytes exhibited partial survival in testis that were administered Mo.
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Fig. 2. Immunohistochemical staining image on the expression of Bax, Bcl-2, caspase-3 and
444
caspase-9 in the testis. (n=30)
445
A, B, C and D represented the expression of Bax, Bcl-2, caspase-3 and caspase-9 in turn;
446
1, 2 and 3 represented the control group, the busulfan group, the busulfan+Mo group in turn;
447
For example: A1 represented the expression of Bax in the control group. Immunohistochemistry
448
demonstrated that Bax, Bcl-2, caspase-3 and caspase-9 protein were not almost expressed in testis
449
(A1, B1, C1 and D1); Bax, Bcl-2, caspase-3 and caspase-9 protein were widely expressed in
450
spermatogenic cells (A2, B2, C2 and D2); Bax, Bcl-2, caspase-3 and caspase-9 protein were
451
weakly expressed in spermatogenic cells (A3, B3, C3 and D3).
ACCEPTED MANUSCRIPT
452 453
Fig. 3. Mo relieved busulfan-induced apoptosis on the expression of Bax, Bcl-2, caspase-3 and
454
caspase-9 in the testis. (n=30)
455
A1: Western blot analysis the expression of Bax, Bcl-2, caspase-3 and caspase-9; A2: Image-J
456
software calculated the gray value of Bax, Bcl-2, caspase-3 and caspase-9.
457
column, values without a common capital superscript differ (P < 0.01). a,b Within a column, values
458
without a common capital superscript differ (P < 0.05). Bus: busulfan Group; Bus+Mo:
459
busulfan+Mo group.
A,B,C
Within each
ACCEPTED MANUSCRIPT In this work, our results indicated Mo at 20 mg/L has a significant recovery effect on the injured testis, and it has obvious therapeutic effect on reproductive dysfunction of infertility mice. The mechanism of Mo therapeutic effect on mice infertility could be due to that Mo could repair the damaged testicular structure, regulate abnormal reproductive endocrine, decrease oxidative stress, and resist cell apoptosis. To our knowledge, this is the first report on the offspring birth to term of Mo treating infertility mice.
ACCEPTED MANUSCRIPT 422 423
Table. 1 Effects of Mo on the organ coefficient, hormones levels, oxidative stress and reproductive parameters in each group male mice. (n=30) Control Organ coefficient
Hormones levels
Oxidative stress
Reproductive parameters
Bus+Mo
Testis
5.501±0.444A
2.643±0.682B
5.563±1.084A
Epididymis
2.478±0.085A
2.259±0.096B
2.459±0.571A
T (ng/L)
12.66±0.90A
10.56±0.28B
13.52±1.29C
FSH (IU/L)
1.07±0.08
1.16±0.11
1.02±0.04
LH (ng/L)
194.89±6.16A
205.18±3.90B
190.40±3.90A
E2(ng/L)
2.82±0.69Aa
2.10±0.10B
2.52±0.11Ab
SOD(U/mgprot)
249.22±22.42A
211.60±14.71B
251.22±31.46A
LDH(U/gprot)
2708.86±123.53Aa
4616.88±129.25B
2818.293±193.21Ab
MDA(nmol/mgprot)
1.02±0.12a
1.19±0.16b
1.03±0.18c
Pregnancy rate
1A (20/20)
0.05B (1/20)
0.95A(19/20)
No. offspring
215A
6B
208A
1:0.97
1:1
1:1.02
10.75A
6B
10.95A
Offspring gender ratio(Male: Female) Average No. offspring 424 425 426 427 428 429 430 431 432
Bus
Data are reported as means (±SD). A,B,C Within a line, values without a common capital superscript differ (P < 0.01) ; a,b,c Within a line, values without a common lowercase superscript differ (P < 0.05). Bus: busulfan group; Bus+Mo: busulfan+Mo group. Control: mice received a single intraperitoneal injection of 50% DMSO at 42 days of age. Bus: mice received a single intraperitoneal injection of busulfan (20 mg/kg body weight) diluted in DMSO at 42 days of age. Bus+Mo: mice received a single intraperitoneal injection of busulfan (20 mg/kg body weight) diluted in DMSO at 42 days of age and were given drinking water containing 20 mg/L Mo at 72 days of age for continuously 42 days. Organ coefficient= wet organ weight/totle wet body weight (mg/g). SOD: superoxide dismutase, LDH: lactate dehydrogenase, MDA: malondialdehyde.