The ameliorative effect of atorvastatin on serum testosterone and testicular oxidant/antioxidant system of HFD-fed male albino rats

Background: There is a mutual effect between central obesity and low total serum testosterone. Moreover, oxidative stress acts as a bridge between obesity and its complications. Taken together, we aimed to evaluate whether atorvastatin (AS), a cholesterol-lowering drug, has protective potential against high fat diet (HFD)-induced low fertility, which was exemplified in serum testosterone determination. Moreover, we aimed to deduce a putative mechanism of action through evaluation of the testicular oxidant/antioxidant system. Methods: Adult male albino Wistar rats ( Rattus norvegicus albinus) were divided into three groups: 1) normal control group, rats were fed a normal diet for four weeks; 2) HFD group, rats were fed an HFD for four weeks; and 3) AS group, rats were fed an HFD and 5 mg/kg/day atorvastatin for the last two weeks of the experiment. Serum atherogenic index, testosterone, and thyroid stimulating hormone were estimated. Moreover, testicular reduced glutathione and malondialdehyde contents, as well as glutathione-S-transferase, superoxide dismutase, and glutathione reductase activities were also determined. The statistical differences were analyzed using analysis of variance (ANOVA). Results: AS ameliorated the increased level of serum atherogenic index induced by an HFD, as well as testicular malonaldehyde and reduced glutathione levels. On the other hand, AS increased the depleted level and activity of serum testosterone and testicular glutathione reductase, respectively, induced by HFD. Conclusion: The ameliorative effect of AS on the deteriorated level of total serum testosterone induced by HFD might partially be due to oxidant/antioxidant disturbance. Further studies should be carried out to evaluate mTOR pathway contribution, which could enable researchers to deduce drugs targeting members of the oxidant/antioxidant system and/or mTOR pathway to ameliorate putative HFD-induced low fertility.


Introduction
Obesity or overweight is related to an elevated risk of many medical problems such as depression and infertility. In those that are obese or overweight, hyperlipidemia can increase the cholesterol content of platelets, endothelial cells, smooth muscle cells, neutrophils and polymorphonuclear leucocytes and may be a source of reactive oxygen species (ROS) 1 . Moreover, increased free fatty acidemia and triglyceridemia, which are consequences of high fat diet (HFD) supplementation, are implicated in obesity complications 2,3 .
The WHO has reported that approximately 1.9 billion people are overweight and 650 million people are obese worldwide 4 . Moreover, defective sperm quality constitutes 30% of infertile couples, who represent around 16% of the worldwide population 5 . Interestingly, no precise cause can be elucidated for primary or secondary infertility in around 25% of couples 6 . Although literature connecting semen parameters and obesity are contradictory, it is thought that there is a mutual effect between low serum testosterone and central obesity 7 . It has been reported that an increased glycerol level, which is HFD-induced, might lead to a leaky blood-testis barrier, disrupting the homeostasis of the tubular fluid and promoting the apoptosis of germ cells 8 .
It has also been suggested that oxidative stress acts as a bridge between obesity and its complications 9 . Dyslipidemiagenerated elevated ROS and/or inflammatory substances are considered to be causes of metabolic disorders such as infertility 10,11 . High ROS levels may render testicular cells ineffective at maintaining a convenient environment for spermatogenesis 12 . Hence, decreasing dyslipidemia may improve HFD-induced poor fertility and atorvastatin (AS), which is a cholesterol-lowering drug, may be beneficial in this way.

Ethical statement
All animal handling was in accordance with the instructions and guidelines of the Experimental Animal Ethics Committee, Faculty of Science, Beni-Suef University, Egypt (Approval Number: BSU/FS/2020/18).

Experimental animals
A total of 30 apparently healthy adult male albino Wistar rats that initially weighed approximately 85 g were used in the beginning of this study to overcome any sudden death or diseases to rats. Later, we excluded seven rats because they did not achieve serum atherogenic index (AX) > 0.24, required for inclusion. Two rats died after sub-allocation of HFD-fed rats. Table 1 shows the final number of rats used and their allocation. The animals were purchased from Helwan University, Cairo, Egypt and then housed in the Biochemistry Department, Faculty of Science at Beni-Suef University. The rats were acclimatized for one week to exclude any intercurrent infection. Rats were kept in polypropylene cages with well aerated stainless-steel covers in a controlled environment that was maintained under a 12-hour light/dark cycle, a temperature of 24°C (±3°C) and 50-70% humidity. The rats were supplied with a standard diet and had free access to tap water. All efforts were made to reduce the number and suffering of the rats under investigation. These efforts included: 1) continuous checking of animal house air conditioning and continuous and careful supply of drinking water; 2) refreshing the animal house with ordinary air daily for 30 mins; 3) cleaning the cages and the animal house; 4) using the effective anesthesia dose; 5) carrying out euthanasia of animals far away from the other alive rats; 6) carefully disposing of euthanized animals far away from the other alive rats.

Materials
Normal standard food was purchased from Mecca Factory, Beni-Suef, Egypt. The high fat diet consists of the normal rat chow mixed with 15% palm oil and 1% cholesterol in accordance with Matos et al., with some modifications 13 . Compositions of normal standard and high fat foods are illustrated in Table 2. AS was purchased from Egyptian International Pharmaceutical Industries Co (EIPCO) 10th of Ramadan City, Egypt. All other chemicals were of analytical grade and were obtained from standard commercial suppliers.
Animal grouping and experimental procedure Rats were divided into three groups as shown in Table 1. Selection of animals was carried out haphazardly without performing any prior randomization method.
1. Normal control group (CN): Rats were fed a normal diet, ad libitum, for four weeks; the whole period of the experiment. Rats were also given the equivalent volume of corn oil, as a vehicle, by oral gavage daily for the last two weeks. 2. HFD group: Rats were fed an HFD, ad libitum, for four weeks of the experiment, in addition to equivalent volume of corn oil by oral gavage daily for the last two weeks.
3. AS group: Rats in this group were fed a high fat diet for four weeks and treated orally with a dose of 5 mg/kg/day atorvastatin suspended in corn oil for the last two weeks of the experiment. The dose was double of that used by Oktem et al. 14 .
The 30 rats were allocated to six cages; five rats/each cage. On the first day, we selected a cage for commencing treatment randomly, then, every day we selected the starting cage serially. Selection of an animal inside each cage was arbitrary. The oral gavage started at 10 AM until nearly 12 AM. Rats treatments were carried out at animal house in Faculty of Science. The route of AS administration was by oral gavage, which is easy and suitable for rats.
The first author was aware of most stages of the experiments. Most of the authors and technicians contributed to animal euthanasia and collection of specimens. Therefore, blinding was not achieved during allocation nor treating stages; however, it was achieved during assessment and data analysis stages.
The dose was adjusted every week based on the changes in body weight (b. w.) to ensure a constant dose per kg b. w. of rats over the entire period of study. After two weeks of the onset of the experiment, rats were fasted overnight (10-12 h) and blood samples were collected from the lateral tail vein for screening of the AX according to the formula: log10 (TG/HDL-c). Rats that exhibited AX less than 0.24 were excluded. This criterion was defined before any data was collected. Rats with AX more than 0.24 were allocated to the HFD group and AS-administered group for an extra two weeks. At the end of the experiment, the rats were deprived of food overnight and anaesthetized by inhalation with diethyl ether. Blood samples were collected from the jugular vein and then the rats were killed by sudden cervical decapitation while they were anaesthetized.
Tissue sampling for biochemical tests Immediately after weighing the testes, each testis was homogenized in 5 ml cold buffer (100mM potassium phosphate, PH 7.0, containing 2mM EDTA) per gram of tissue. For the biochemical analysis of antioxidants, including reduced glutathione (GSH) levels were determined according to the method of Beutler et al. 15 . Moreover, glutathione-S-transferase (GST), superoxide dismutase (SOD), and glutathione reductase (GR) activities were also determined [16][17][18] . Besides that, determination of malondialdehyde (MDA) level, as an indicator for lipid peroxidation, was also estimated according to Ohkawa et al. 19 .
Determination of testicular GSH content. The protein content of the testicular sample was precipitated by adding an equal volume of 4 % sulphosalicylic acid. After centrifugation, 0.5 ml of the supernatant was added to 4.5 ml of 0.1 mM Bis-(3-carboxy-4-nitrophenyl)-disulfide reagent in 0.1 M phosphate buffer, pH 8. The color intensity was determined spectrophotometrically at 412 nm.

Determination of testicular GST activity.
After addition of glutathione (0.5 mM), and sodium phosphate buffer (0.1 M), pH 7.3, to the tissue sample, preincubation was carried out for 5 min at 37°C. Then, 1-chloro-2,4-dinitrobenzene (CDNB, 0.5 mM) was added to the incubation mixture. Thereafter, trichloroacetic acid solution (33%) was added and the mixture was centrifuged. The CDNB conjugate was measured in the supernatant at 340 nm. The quantity of enzyme that enhanced the formation of 1µmol of CDNB conjugate/mg protein/min was considered as one unit of enzyme activity. The molar extinction coefficient was 9.6 mM−1 cm−1.

Determination of testicular SOD activity.
The reaction mixture contained the sample, tris/EDTA buffer, 12.1%, pH 8, and pyrogallol 10 mM. SOD prevents pyrogallol auto-oxidation by removing superoxide anions. As described, 0.1 buffer was mixed with 1 ml tissue sample, then 0.05 ml pyrogallol was added. The absorbency was read at 0 and 10 mins after the addition of pyrogallol. One unit of enzyme activity is the amount of enzyme that resist a 50% change in extinction per one min. The modification used to determine SOD activity was the use of pyrogallol instead of cytochrome c. ΔA/min was calculated as follows: Enzyme activity (U/ml) = Then U/ml was converted to U/g tissue.
Determination of testicular MDA content. 1 ml of tissue homogenate was mixed with 2 ml of trichloroacetic acid (TCA, 7.5%) and then centrifuged. 2 ml of the formed supernatant was mixed with thiobarpeturic acid (TBA, 0.7 %) and boiled for 10 mins. The color intensity of the reactants (TBARS) was measured at 532 nm. An extinction coefficient of 156000 M−1 cm−1 was used for calculation.

Determination of serum HDL-cholesterol concentration.
Serum HDL-cholesterol concentration was measured using the Randox HDL-cholesterol assay (Cat No. CH 203). Briefly, 50 µl of phosphtungestic acid (13.9 mol/l) was added to 500 µl of sample to precipitate LDL, VLDL and chylomicron fractions in the presence of magnesium ions, allowed to stand for 10 min, centrifuged for 10 min at 4000 rpm. Then, the supernatant was collected to determine the cholesterol content as described before. HDL-cholesterol concentration was calculated as follows: HDL-cholesterol concentration (mg/dl) = A sample × 180.

Determination of serum total testosterone level.
Serum total testosterone level was measured using the VIDAS® Testosterone II kit (Cat No. 414320, bioMérieux, France). The antibodies used were monoclonal anti-testosterone antibodies produced in mice. All assay steps were performed automatically by the instrument. The strip consisted of 10 wells covered with a labeled foil seal. The last well of each strip was a cuvette in which the fluorometric reading was performed. The wells in the center section of the strip contained the various reagents required for the assay.
Briefly, the procedure aimed, firstly, to isolate testosterone from the carrier proteins in the sample by using the pre-treatment solution. The pre-treated sample was transferred to a well containing a testosterone antibody (conjugate) labelled with alkaline phosphatase. For the anti-testosterone-specific antibody sites, the antigen in the sample and the testosterone antigen attached to the SPR ® inner wall. During the washing steps, unbound pieces were removed. The substrate (4-Methylumbelliferyl phosphate) was cycled in and out of the SPR ® during the final detection stage. The hydrolysis of this substrate was catalyzed by the conjugate enzyme into a fluorescent product (4-Methylumbelliferone), whose fluorescence was measured at 450 nm. The fluorescence intensity was inversely proportional to the testosterone concentration present in the sample. The results were automatically determined by the instrument at the end of the assay with regard to the calibration curve stored in memory.

Determination of serum TSH level.
Serum TSH level was measured using the VIDAS® TSH kit (Cat No. 30400, bioMérieux, France). The protocol was like that of total testosterone determination except we used mouse monoclonal anti-TSH antibodies instead of anti-testosterone antibodies.

Statistical analysis
Data are expressed as mean ± SE. The statistical differences between continuous variables was analyzed using analysis of variance (ANOVA). Statistical analysis was performed using SPSS v.24 (SPSS Inc., released 2007, SPSS for Windows, version 16.0, Chicago, IL). Values with p < 0.05 were considered significant.

Results
Data resulting from our experiment were deposited in Harvard Dataverse 23 . Supplementing rats with an HFD produced a significant increase (p < 0.05) of 31% in serum AX relative to CN. However, administering AS ameliorated this effect by significantly decreasing serum AX by 18% relative to the HFD-fed group. HFD caused a profound decrease of 62% in total serum testosterone, while AS alleviated this negative effect by increasing the total serum testosterone level by 92% (p < 0.05). However, neither HFD nor AS showed a significant effect on serum TSH at p ≥ 0.05 (Table 3).
Regarding MDA, HFD exerted a tremendous increase in testicular levels of MDA of 113%. AS exhibited an amelioration of this effect, causing a 54% decrease in MDA levels relative to the HFD group (p < 0.05). Testicular GSH content was profoundly increased by HFD supplementation by 87%, while AS supplementation caused a 30% decrease in GSH content (p < 0.05) ( Table 4).
GR activity was decreased by 42% by HFD but increased by 112% by AS (p < 0.05). There were moderate increases of 14% and 11% in SOD and GST activities, respectively, after HFD supplementation. However, a decrease of 4% was observed in SOD activity after AS administration (p < 0.05), while AS produced an insignificant change in GST activity relative to the HFD group (p ≥ 0.05) ( Table 5).

Discussion
Lipid profile amelioration is considered to be an important target in order to foster β-oxidation of fatty acids, otherwise fatty acids would be oriented towards the esterification pathway, leading to accumulation of TAG and LDL cholesterol 24 .
Hence, statins such as AS can help in this trend. They can up-regulate the LDL receptor and reduce cholesterol absorption and biosynthesis, an effect that leads to rapid clearance of LDL particles from circulation 25 .
Statins were thought to improve sperm parameters regarding dyslipidemia. However, Pons-Rejraji et al. counteracted this finding, as statins might induce local inflammation or oxidative stress, with persistent effects on prostatic and epididymal secretion 26 . In agreement with this, some evidence has shown that statins might be detrimental to testosterone production 27 and might induce impotence 28 . Other studies indicated that patients undergoing statin therapy to lower serum cholesterol levels have neither androgen deterioration 29 nor impotence 30 due to sterol regulatory element binding protein (SREBP)/ Srd5a2 system alteration 31 . The different pharmacokinetic or physicochemical properties of various statins may contribute considerably to total serum testosterone levels.  To share in this debate, we introduced an HFD to rats and treated them with AS. In our experiment, an HFD profoundly decreased serum testosterone, which was counteracted by supplementing with AS, although serum levels were not elevated to that of the normal control. It was previously reported that the decrease of total serum testosterone in obese men is due to increased serum estrogen as a result of increased aromatase activity 32 .
The detrimental effects of HFD may be mediated, in our study, via an elevated testicular malondialdehyde level, which is a marker of oxidative stress. However, the ameliorative effect of AS may be partially explicated by lowering serum AX and testicular malondialdehyde levels. Surprisingly, HFD markedly elevated testicular glutathione level and GR activity, which may be a kind of compensatory mechanism. The substantial contribution of the oxidant/antioxidant system in the deterioration effects of HFD on fertility was obvious.
Additionally, cholesterol synthesis is not completely inhibited in peripheral tissues in statin-treated patients 33 . Therefore, enough cholesterol is available in gonadal and adrenal cells to maintain steroid hormone synthesis; this in line with our data.
Accumulated data suggest mTOR as a promising mechanism for explaining HFD-caused detrimental effects on fertility.
In one study, mTOR and p70s6k were weakly expressed in the HFD group, which may be reversed by statin 34 .
Our data enables researchers to deduce drugs targeting members of the oxidant/antioxidant system and/or mTOR pathway to ameliorate putative HFD-induced infertility. This project contains the following underlying data:

Data availability
• Stat (AS).docx (statistics report of data) • Outcomes (AS).docx (raw GSH, GR, SOD, and GST and MDA levels, as well as serum total testosterone and TSH levels for all 21 rats) Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

3.
Yes *Is 4 weeks of HFD is enough to produce hypercholesterolemia *Why the author measure TSH, what is the relation to the protocol of the study (reproduction) need to be explain. *The goal of the study is to induce reproductive disorder by HFD and its treatment with Ator *HFD is related to cholesterol and so testosterone. please focus on these point with the role of Ator

Materials and Methods
*Why author choose palm oil instead of beef tallow as source of fat . *The amount of dietary composition, proteins, fat carbohydrate should be provided

Results:
* Table 3 showed non significant role of TSH between group what is the important of its measurement for the study. * Table 4 GSH as antioxidant biomarkers should be decrease in HFD group, however in the table it increases, need to be revised. *How HFD decrease the level of TT , since HFD increase the serum cholesterol *Fertility parameters required to be focus on specially Sperm count etc.

Discussion:
-How Ator which is lowering cholesterol medication act as antioxidant, what is the mechanism, is it related to lower cholesterol. Please Provide reference and mechanism.

Minor comment
Did the HFD group continue on HF feeding until the end of the experiment (last 2 weeks)