Effect Of Cistanche Tubulosa Extracts On Male Reproductive Function in Streptozotocin–Nicotinamide-Induced Diabetic Rats-Ⅰ

1. Introduction 

Diabetes mellitus (DM) is a condition in which there are increased glucose levels in the blood due to the inefficiency of the pancreas in producing enough insulin, or the inability of the body to use it effectively. According to the WHO, the number of people with diabetes rose from 108 million in 1980 to 422 million in 2014 [1]. There are four main categories: pre-diabetes (a stage before diabetes), type 1 (where the pancreas fails to produce insulin), type 2 (the body fails to use insulin), and gestational diabetes (which occurs during pregnancy). Oxidative stress occurs due to the imbalance between the production of reactive oxygen species (ROS) and antioxidants [2] finally leading to the diabetic condition. The complications of diabetes include failure of the kidney, nerve damage, blindness, stroke, heart attack, fetal death, and infertility. [1]. Studies show that sperm nuclei, deoxyribonucleic acid (DNA), and mitochondria were significantly damaged in male diabetic patients [3]. Oxidative stress during the transportation of sperm will alter the process of the male reproductive system [4,5]. 

NATURAL CISTANCHE TUBULOSA FOR IMPROVING SEXUAL FUNCTION PHGS75% ECH 30% ACT 12%

The process of spermatogenesis is controlled by the hypothalamus–pituitary–gonadal (HPG) axis. The gonadotropin-releasing hormone (GnRH) produced by the hypothalamus stimulates the production of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Leydig cells are found adjacent to the seminiferous tubule and produce testosterone under the control of LH. FSH triggers the production of antigen-binding protein (ABP) that helps to pass the male hormone. Thus, infertility and other problems mainly arise due to the disturbances that occur in the HPG axis. Administration of the combination of streptozotocin–nicotinamide induces diabetes in experimental rats. Streptozotocin is a chemical compound toxic to insulin-producing β cells in the pancreas and nicotinamide is a water-soluble vitamin that protects the β cells from complete damage [6]. 

Cistanche tubulosa is a desert plant species also known as "Rou Cong-Rong" [7]. It is a non-chlorophyllic parasitic plant that grows mainly on the root of the Calotropis procera tree and is widely distributed in the arid land of Gansu, Qinghai, Xinjiang, Mongolia, Iran, and India. The common name of Cistanche tubulosa is "Desert Hyacinth". It is widely accepted in Chinese traditional medicine and has been given the name "Ginseng of the Desert". It is widely used in the medicinal field to treat morbid leucorrhea, profuse metrorrhagia, chronic renal diseases, constipation, impotence, and infertility. Chemical constituents of Cistanche tubulosa consist of non-volatile phenylethanoid glycosides (PHGs), iridoids, lignans, volatile oils, alditols, oligosaccharides, and polysaccharides [8]. Studies show that echinacoside from this herb protects the damaged fibroblast by regulating ROS levels [9]. This compound produces antioxidant, vasorelaxation, and anti-inflammatory activities together with neuroprotection and osteoporosis prevention [10,11]. 

This study aimed to investigate the effect of Cistanche tubulosa extract containing ECH on the reproductive dysfunction of the streptozotocin–nicotinamide-induced diabetic rat.

2. Materials and Methods 

2.1. Materials 

ECH and CTE were purchased from Sinphar Pharmaceutical Co., Ltd (Yilan, Taiwan). Advanced glycation end products (AGEs) were purchased from Biovision (San Francisco, CA, USA). LC-540 and TM3 cell lines were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Five-week-old Sprague-Dawley (SD) male rats were purchased from the National Laboratory Animal Center (Taipei, Taiwan). Feed Lab Diet® was purchased from PMI Nutrition International, Inc. (Taipei, Taiwan). 2,2-diphenyl-1-picrylhydrazyl (DPPH) was obtained from Sigma (St. Louis, MO, USA). Methanol and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were also purchased from Sigma. Dulbecco's modified Eagle medium/F12 and trypsin-EDTA were obtained from GIBCO (New York, NY, USA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), dichloro-dihydro-fluorescein diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), and nitroblue tetrazolium (NBT) were purchased from Sigma. Glucose enzymatic kits were obtained from Kyokutoseiyaku, Tokyo, Japan. Insulin ELISA kits were purchased from Mercodia AB Inc., Sylveniusgatan 8A (Uppsala, Sweden). T-PER Tissue Protein Extraction Reagent was acquired from Thermo Scientific (Chicago, IL, USA). Anti-human/Mouse/Rat nuclear factor-kappa B NF-κB polyclonal antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Anti-Mouse/Rat receptor for advanced glycation end products (RAGE) polyclonal antibodies, anti-Human/Mouse/Rat StAR polyclonal antibodies, and anti-Human/Mouse/Rat Cytochrome P450 17A1 monoclonal antibodies were purchased from Gene Tex (Irvine, CA, USA). Anti-Human/Mouse/Rat CYP11A1 polyclonal antibodies were obtained from Cell Signaling Technology (Beverly, PA, USA). Easy-Blue reagent was purchased from Invitrogen, Thermo Fisher Scientific (Carlsbad, CA, USA). Agarose and DNA marker 100 bp were obtained from Promega, Corporation (Madison, WI, USA). RNeasy Lipid Tissue Mini Kit was purchased from QIAGEN, Hilden, Germany.

NATURAL CISTANCHE TUBULOSA FOR IMPROVING SEXUAL FUNCTION PHGS75% ECH 30% ACT 12%

2.2. Methods 

2.2.1. In Vitro Analysis 

LC-540 and TM3 Cell Culture: The LC-540 cell lines were cultured at 37 ◦C in Earle's Balanced Salt Solution (EBSS) medium supplemented with sodium bicarbonate (1.5 g/L), L-glutamine (2 mM), non-essential amino acids (0.1 mM), sodium pyruvate (1.0 mM), and fetal bovine serum (FBS) (10%) in a 5% CO2 incubator (CO2 incubator, Napco 5410, Taiwan). The TM3 cell line was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with glucose (4.5 g/L), sodium pyruvate (0.5 mM), L-glutamine (2.5 mM), sodium bicarbonate (1.2 g/L), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 15 mM), and fetal calf serum (FCS, 10%) or horse serum (5%) at 37 ◦C in a 5% CO2 incubator.

Determination of the Cell Viability of Echinacoside (ECH): 3-(4, 5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) reagent was used for cell viability assay. The cell concentration was adjusted to 2 × 105 cells / mL in a 96-well plate. Then, 20 µL of ECH (diluted in 2% of medium) was added and incubated for 24 h at 37 ◦C in a 5% CO2 incubator. Later, 100 µL of MTT reagent was added and incubated for 4 h at 37 ◦C under dark conditions. The absorbance was measured at 570 nm (ELISA reader, Dynatech MR5000, Kloten, Switzerland). 

Relative cell viability (%) = [(Asample at 570 nm − Ablank at 570 nm)]/[(Acontrol at 570 − Ablank at 570)] × 100.

Nitroblue Tetrazolium (NBT) Reduction Assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) Assay: The number of cells was adjusted to 1× 106 cells/mL. Then, 50 mg/mL of ECH, resveratrol (RES), and advanced glycation end-products (AGEs) (control) were added and co-cultured for 18 h at 37 ◦C. Then, 0.3 mL of NBT solution (0.1 mg/mL NBT, 5% FBS, and 3% dimethyl sulfoxide (DMSO) dissolved in 10 mL DMEM) was added and incubated at 37 ◦C in 5% CO2 for 1 h. After centrifugation (at 800× g for 3 min) the supernatant was removed and 200 µL DMSO was added and shaken for 5 min in an ultrasonic oscillator (Delta Ultrasonic Cleaner D 200, Keelung, Taiwan). The absorbance was measured at 630 nm. For DPPH radical assay, Trolox (in 95% alcohol) was taken as the standard. Then, 75 µL of 0.5 mM DPPH solution was mixed with 25 µL of samples and allowed to react at room temperature in a dark place for 30 min. The mixture was shaken and the absorbance was measured at 517 nm.

Reactive Oxygen Species (ROS) Content Analysis: The cell number was adjusted to 2 × 105 cells/mL in a 12-well plate containing 2% FBS. Then, 50 µL/mL of ECH, RES, and AGEs were added to the plate and incubated at 37 ◦C for 24 h. After 24 h, 20,70 -dichlorofluorescein-diacetate (DCFH-DA) was added and incubated at 37 ◦C for 30 min. After centrifugation (400× g, 5 min), the supernatant was removed and the cells were washed twice with phosphate-buffered saline (PBS). After, cells were suspended in 1 mL of PBS and the levels of ROS were determined by using a flow cytometer (Flow Cytometer, Becton Dickinson, CA, USA). 

Identification and Quantitative Analysis of Protein: Proteins were extracted by using a protein extraction reagent (T-PER). The cell density was adjusted to 2 × 105 cells/mL in a 12-well plate and 50 µL/mL of ECH, RES, and a receptor for the advanced glycation endproduct (RAGE) antagonist, and AGEs were added and incubated at 37 ◦C in a 5% CO2 incubator for 24 h. After, 400 µL of T-PER were added, scraped off the cells, and centrifuged at 125,000× g for 20 min (4 ◦C). The supernatant was collected and stored at −80 ◦C (−80 ◦C freezer, Nuaire, Plymouth, MN, USA) for further analysis. A bicinchoninic acid (BCA) kit was used for protein quantification. Then, 10 µL of standard cell solution and 200 µL of BCA reagent were added to 8-well plates and incubated at 37 ◦C in a 5% CO2 incubator for 30 min. The absorbance was measured at 562 nm. Protein identification analysis was performed by sodium dodecyl sulfate (SDS)-polyacrylamide gel (SDS-PAGE) electrophoresis. Samples were prepared by adding protein lysate to a protein loading buffer and western blot analysis was performed to detect the specific proteins.

2.2.2. In Vivo Analysis 

Animal Model Design: Sixty 4-week-old male Sprague–Dawley (SD) rats were purchased from the National Laboratory Animal Center (Taipei, Taiwan). Each rat was housed individually in disinfectant stainless steel cages under controlled temperature (23 ± 1 ◦C) and humidity (40–60%) with a 12 h light/12 h dark cycle. Food and water were provided ad libitum. Each rat was domesticated in the first week and given laboratory rodent diet 5001 as the main diet. All procedures followed the standard of the Institutional Animal Care and Use Committee (IACUC Approval No. 101026) of the National Taiwan Ocean University, Taiwan. After domestication, rats were divided into two groups: the control group (Con) which was fed lab diet 5001, and the diabetic group which was fed a high-fat diet (HFD, 40%) for the entire experiment. After being fed with the HFD for 4 weeks, the diabetic group rats were injected with streptozotocin (65 mg/kg) (STZ) to induce diabetes mellitus (DM). Within 15 min of injecting STZ, nicotinamide (230 mg/Kg Body Weight) was injected. A week after injection, an oral glucose tolerance test (OGTT) was conducted to determine the successful induction of diabetic mellitus (DM). After that, the animals were divided into six groups: the control (fed with lab diet 5001); DM group (DM + 45% HFD); DMR group (DM + rosiglitazone: 0.571 mg/kg BW) + 45% HFD; DME1 group (DM + CTE: 80 mg/kg BW) + 45% HFD; DME2 group (DM + CTE: 160 mg/kg BW) + 45% HFD; and DME4 group (DM + CTE: 320 mg/kg BW) + 45% HFD. Rosiglitazone (RSG) was taken as the positive control. Three doses of CTE (80, 160, and 320 mg/kg) were used according to the recommendation of the Taiwan Food and Drug Administration (TFDA) health food functional evaluation guidelines. Treatments were given until the end of the experiment. All experimental rats were sacrificed after 6 weeks. The blood collected from the rats was centrifuged at 3000 rpm for 15 min at 4 ◦C. The supernatant was examined for the following analysis:

Total Glucose, Triglyceride, and Cholesterol Determination: The glucose determination was carried out by glucose enzymatic kits. Then, 20 µL of blood plasma samples were added to the reagents and kept at 37 ◦C for 5 min. The total triglycerides and total cholesterol concentration were determined by using triglyceride and cholesterol enzymatic kits. Then, 10 µL of blood plasma was added to the reagents and the experiment was conducted according to the manufacturer's instructions. The absorbance was measured at 510 nm. 

Plasma total glucose/triglyceride/cholesterol (mg/dL) = (Asample − ABlank)/(Astandard − ABlank) × 200. 

Asample: Absorbance of blood samples, ABlank: Absorbance value of kits without a sample, Astandard: Absorbance value of the standard reagent, 200: standard reagents at a concentration of 200 mg/dL.

Insulin, Leptin, and Homeostatic Model Assessment–Insulin Resistance (HOMA-IR) Determination: The insulin level was determined by insulin ELISA kits. Here, 25 µL of blood plasma was added to the reagents, and the absorbance was measured at 450 nm. Total leptin content was determined by using a leptin enzyme immunometric assay kit. Then, 100 µL of plasma was analyzed and the absorbance was measured at 450 nm using an ELISA reader. The whole experiment was conducted according to the manufacturer's instructions. The HOMA-IR value was determined from the homeostasis model assessment equation = Fasting plasma insulin concentration (mg/mL) × fasting plasma glucose concentrations (mmol/L)/22.5.

Plasma Lipid Peroxidation: Here, 0.5 mL of blood plasma was mixed with 1 mL of reagent (15%, w/v trichloroacetic acid in 0.25 N hydrochloric acid (HCl) and 0.375%, w/v thiobarbituric acid in 0.25 N HCl) and placed in a water bath (Water Bath, BUCHI 461, Zürich, Switzerland) at 100 ◦C for 15 min. After cooling, 1 mL of n-butanol was added, shaken vigorously, and centrifuged at 1500× g for 10 min. The supernatant was collected and the absorbance was measured at 532 nm [12]. 

Malondialdehyde (MDA) concentration (nM/mL) = [(A sample at 532 nm − Ablank at 532 nm)/ (A standard at 532 nm − Ablank at 532 nm)] × 5.

standard at 532 nm − Ablank at 532 nm)] × 5. Determination of Plasma Testosterone and Luteinizing Hormone (LH) Levels: The testosterone ELISA kit was used to measure the level of testosterone in the blood plasma. RIA kit was used to determine the LH concentration. Then, 50 µL of plasma was added and the calculations were based on the concentration of the hormone LH-RP-3 (standard). The further steps were conducted according to the manufacturer's instructions. 

Determination of Tumor Necrosis Factor (TNF)-α and Interleukin-6 (IL-6) Concentration: The captured antibody was diluted 250 times in coating buffer, added to a 96-well plate (100 µL/well), and kept at 4 ◦C for overnight. The supernatant was aspirated and washed five times with washing buffer (1 time PBS, 0.05% Tween 20). After incubation (1 h) with 200 µg of assay diluent, the cells were washed 5 times with washing buffer and 100 µL of TNF-α standard solution or test samples were added to each well and incubated for 2 h. After washing, 100 µL of IL-6-detected antibodies were added and incubated for 1 h at room temperature. The supernatant was aspirated and washed 5 times. Then, 480 µL of enzyme avidin-horseradish peroxidase (HRP) was added and incubated for 30 min at room temperature. The supernatant was aspirated and washed five times with a washing buffer. Then, 100 µL of substrate solution was added and incubated for 15 min at room temperature. Later, 50 µL of stop solution (1M phosphoric acid) was added to each well and the absorbance was measured at 450 nm.

NATURAL CISTANCHE TUBULOSA FOR REPAIRING SEXUAL FUNCTION PHGS75% ECH 30% ACT 12%

Analysis of Sperm and Testis Parameters

Sperm Sample Collection: The sperm sample collection was performed according to the method previously reported in [13]. The sperm were collected from the supernatant and used for further analysis. 

The Number of Sperm, Sperm Motility, and Abnormal Sperm: The number of sperm was calculated using the hemocytometer and trypan blue solution. Then, 100 µL of sperm liquid was mixed with trypan blue solution and the number of sperm, motility, and abnormal sperm were calculated using a hemocytometer and microscope [14].

Nitro blue Tetrazolium NBT Reduction and Lipid Peroxidation of Sperm and Testis: The lipid peroxidation and NBT reduction were analyzed according to the same procedure of plasma analysis.

Determination of Superoxide Dismutase (SOD) and Catalase Activity: Superoxide dismutase (SOD) activity was analyzed by using the RANSEL kit. Here, 0.05 mL of testicular homogenates were added to 1.7 mL of reaction solution (0.05 mM xanthine, 0.025 mM 2-(4-iodophenyl)- 3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT)). After mixing, 0.25 mL xanthine oxidase (80 U/L) was added and reacted at room temperature for 30 s. The absorbance was measured at 505 nm. The protein concentration of the testicular homogenate was determined by Bio-Rad DC protein assay kit and its specific activity (U/mg protein) was calculated. The catalase (CAT) activity was determined according to a previously reported method [15]. 

H&E (Hematoxylin and Eosin) Staining 

The testis was soaked in 10% of formalin for 24 h and stored at 4 ◦C. The tissues were in the micro size and attached to the slide. After fixation (95% methanol + 5% acetic acid), the slides were immersed in hematoxylin for 3 min, washed with running water for 5 min, and then soaked them with 50%, 70%, and 90% alcohol for one minute. After that, the slides were stained with eosin for 10 s and soaked in 100% alcohol for one minute until it faded. Finally, the slide was soaked in xylene for one minute. Later, the slide was air-dried and sealed. The stained tube was placed in an inverted phase contrast microscope (Inverted Phase Difference Microscope, Olympus CK-2, Tokyo, Japan) to observe the morphology. 

Analysis of the Hypothalamus 

The mouse KISS1, G-protein coupled receptor (GPR) 54, suppressor of cytokine signaling 3 (SOCS-3), and sirtuin 1(SIRT1) gene sequences were identified from the NCBI (National Center for Biotechnology Information) gene database and a specific primer was designed using Primer 5.0 PREMIER Biosoft, Palo Alto, CA, USA.). The primers used in the experiment are listed in Table 1.

Extraction of Ribonucleic acid (RNA) from the Hypothalamus. The extraction of RNA from the hypothalamus of the brain was carried out by using the RNeasy Lipid Tissue Mini Kit. The obtained mRNAs were quantitatively measured by reverse transcription (RT) and polymerase chain reaction (PCR). The compliment DNA obtained from RT analysis was stored at −20 ◦C for later use. The extracted DNA was subjected to polymerase chain reaction (PCR) analysis using Taq polymerase. Then, 5~10 µL of the PCR product was taken and analyzed by electrophoresis on a 1.5% agar colloid. The image was taken in a UVP BioDoc-It imaging system. Reverse-transcribed complementary deoxyribonucleic acid (cDNA)was taken and PCR was performed using the IQ SYBR Green Supermix Kit. Later, it was analyzed with iQTM 5 Optical System Software. Proteins were extracted by using a protein extraction reagent (T-PER). Then, 20 mg of testicular homogenate was added to 400 µL of T-PER and centrifuged (High-Speed Centrifuge, Hettich CR-12, Tuttlingen, Germany) at 4 ◦C (125,000× g) for 20 min. The following method was similar to" identification and quantification analysis of protein" in in vitro analysis. 

2.3. Statistical Analysis 

The experimental results were expressed as the mean ± standard deviation (Mean ± SD) and the data were analyzed using Statistical Product & Service Solutions (SPSS) 11.0 software, IBM, Armonk, New York, NY, USA. The differences between the groups were analyzed by one-way analysis of variance (ANOVA). Multiple comparisons of different groups were analyzed by Duncan's test at the value of p < 0.05 as the significant level. 

3. Results 

3.1. In Vitro Analysis 

3.1.1. Comparison of Antioxidant Activities of ECH, CTE, and RES 

Long-term high oxidative stress and chronic inflammation are the main causes of diabetic complications [16]. CTEs contain various phenylethanoid glycosides such as echinacoside (ECH) and acteoside, with the potential for antioxidant activity [17]. DPPH radical scavenging assay was conducted to evaluate the antioxidant activity of ECH. Trolox and resveratrol (3,5,4'-trihydroxystilbene, RES) were taken as the standard and positive control respectively. As shown in Figure 1, ECH showed better radical scavenging activity and the activity was significantly higher than that of the positive control (RES).

3.1.2. Cell Viability of ECH on LC-540 and TM3 Leydig Cells

MTT assay was performed to evaluate the cell viability of different concentrations of ECH. LC-540 Leydig cells and TM3 Leydig cells showed more than 80% of viability after being treated with ECH (Figure 2). As compared to the other concentrations, a 100-µM concentration (dilutions of 100 µM in 2% FBS) of ECH showed better cell viability in TM3 Leydig cells. The result indicated that the increased concentration of ECH does not contribute any significant toxicity to the cells. 

3.1.3. Effect of ECH on AGE-Induced Superoxide Production by NBT Assay in LC-540 and TM3 Leydig Cells

 AGEs cause oxidative damage in the body due to the oxidation of glucose and the formation of free radicals such as O2-, hydroxyl radicals, and carbonyl groups [18]. After being stimulated with 50 μg/mL of AGEs, the LC-540 (Figure 3a) and TM3 (Figure 3b) cells were treated with ECH and RES (5 μM and 10 μM each). The results showed that the production of superoxide anion was increased in the control group (stimulated AGEs), but the production of superoxide anion was decreased after the control group (stimulated AGEs), but the production of superoxide anion decreased after of superoxide production and protected the cells from oxidative damage. In the case of LC-540 cells, 10 µM ECH showed almost similar activity with the normal group. The superoxide production in both cells was decreased with an increase in the concentration of both ECH and RES. 

NATURAL CISTANCHE TUBULOSA FOR IMPROVING SEXUAL FUNCTION PHGS75% ECH 30% ACT 12%

3.1.4. Effect of ECH on H2O2 Production in AGE-Stimulated LC-540 Leydig Cells 

DCFH-DA assay was executed to detect the presence of oxidative species. After entering into the cells, the fluorescent dye DCFH-DA is oxidized by intracellular H2O2 and forms dichlorofluorescein (DCF). As shown in Figure 4, there was a significant increase in intracellular H2O2 production in the AGE-stimulated group (control) whereas the addition of 10 µM ECH and RES significantly reduced the H2O2 production in cells. Administration of 10 µM ECH resulted in only about 47.1% of H2O2 production, significantly lower than in the control group.

3.1.5. Effect of ECH on RAGE and NF-κB Protein Expression Levels in AGE-Stimulated LC-540 Leydig Cells 

Western blot analysis was performed to confirm the presence of RAGE in LC-540 Leydig cells. The effect of ECH on RAGE (Figure 5a) and NF-κB (Figure 5b) protein expression levels in AGEs-stimulated Leydig cells were shown in Figure 5. The results showed that 50 µg/mL concentration of AGEs induced higher RAGE and NF-κB expression in LC-540 Leydig cells and 10 µM concentration of ECH and RES significantly reduced the expression of RAGE and NF-κB. The expression of NF-κB was significantly lower in RES and ECH than in RAGE antagonists. So, the results confirmed that the ECH reduced the level of inflammation by decreasing the level of RAGE and NF-κB.

3.1.6. Effect of ECH on the Testosterone Synthesis pathway in AGE-Stimulated LC-540 Leydig cells.

The process of spermatogenesis and male infertility depends upon the presence of testosterone. As shown in Figure 6, the expressions of StAR (Figure 6a), CYP11A1 (Figure 6b), CYP17A1 (Figure 6c), and HSD17β3 (Figure 6d) proteins were significantly decreased in AGE-stimulated LC-540 Leydig cells (control group). The expressions of StAR, CYP11A1, CYP17A1, and HSD17β3 proteins were significantly increased when the RAGE antagonist, RES, and ECH were added. The increased expression of StAR and CYP11A1 proteins was seen in both the ECH- and RES-treated groups. The expression level of CYP17A1 was almost similar in the RES and ECH groups. The expressions of the HSD17β3 protein in ECH-treated Leydig cells were much higher than those of the RES and RAGE antagonist-treated cells. Increased expression of StAR, CYP11A1, CYP17A1, and HSD17β3 proteins indicated the normal production of testosterone.