Cannabis and metformin on diabetic male Wistar rat sperm and reproductive organ parameters

Abstract

Purpose

Cannabis use has reportedly increased in type 2 diabetic users as a possible co-treatment for associated pain and inflammation. Both cannabis and metformin (an anti-diabetic drug) have a limited number of studies completed on their effect on male reproductive parameters in a diabetic model. This study determined if cannabis and metformin administration alter various reproductive parameters in diabetic male rats.

Methods

Male Wistar rats (n = 35) were fed on a high fat diet and injected with streptozotocin (30 mg/kg rat) to induce a type-2 diabetic model. Treatment groups received cannabis based on Delta-9-Tetrahydrocannabinol (THC) concentrations of 1.25, 2.5 and 5 mg/kg per rat and metformin (50 mg/kg) every alternate day for 10 weeks. Organ weight; serum testosterone levels and sperm count, motility, lipid peroxidation, citrate synthase and lactate dehydrogenase activities were measured.

Results

Cannabis treatment induced a significant concentration dependent decrease in sperm motility at 5 mg/kg rat THC (P = 0.009) administration. Metformin significantly (P = 0.035) increased sperm counts and lactate dehydrogenase activity (P = 0.002). Both cannabis and metformin negatively affected testosterone concentrations.

Conclusions

Cannabis needs to be used cautiously as an alternative treatment in diabetic males based on the negative effects observed for the various reproductive parameters in this diabetic rat model.

Introduction

Cannabis and its derivatives have systemically started flooding the market as alternative treatment or co-treatments for pre-existing ailments especially in countries with more lax legislation in regards to cannabis use [1, 2]. Several medical therapies have incorporated the use of cannabis as a natural treatment for various neurological and inflammatory disorders [3]. Due to its anti-inflammatory and pain-relieving properties, cannabis has become an alternative treatment used as self-medication for individuals afflicted with obesity and type 2 diabetes [4]. In the United Kingdom an estimated 50 000–100 000 individuals with diabetes use prescription-based cannabis or cannabinoids as supplementary treatment and there is no regulation of dose or frequency of self-prescription [5].

Evidence indicates cannabis can be beneficial when used as a diabetic treatment and a limited number of investigations have been undertaken regarding the effects on male fertility for this chronic disease. Current studies available have linked cannabis usage to a decrease in fertility parameters such as an increase in spermatogenesis oxidative stress, reduced serum testosterone concentrations and developmental anomalies in male reproductive accessory organs with a few focussed on diabetic models [6, 7].

Similar to cannabis, the most commonly prescribed drug for type 2 diabetes, metformin, is limited in its knowledge base in regards to male fertility [6]. Metformin is a biguanide drug administered orally with over 78 million prescriptions administered in the United States in 2017 [8]. Metformin’s mechanism of action is primarily to increasing the sensitization of peripheral tissues to insulin and thereby improve glucose uptake [9]. The research performed on male fertility thus far has indicated some positive results including increase sperm, sperm motility and decreased oxidative stress [6].

A decreased fecundity has been associated with males which suffer from obesity and/or type 2 diabetes and is accompanied by decreased testosterone concentrations, sperm counts and motility while simultaneously influencing epigenetics and DNA integrity [10, 11]. This investigation aims to broaden the knowledge base on both cannabis and metformin use indicating whether these treatments may further aggravate or improve fertility parameters in diabetic male Wistar rats.

Materials and methods

Cannabis analysis and treatment preparation

Cannabis resin (Cannabis sativa) was prepared from dried plant material collected from the local authorities (permit number: POS 026/2016/2017). Extraction and quantification was performed in accordance to the protocol established by Ramlugon et al., (2018) [12]. Plant material was dried, crushed and extracted using chloroform. The extract was then filtered and concentrated using rotor evaporation. Final concentration was performed under a constant atmosphere of nitrogen. Major cannabinoid constituents were quantified using reverse phase high performance liquid chromatography. The THC, CBN and CBD ratio was determined to be 5 : 2 : 1 respectively. Cannabis resin was diluted to three treating concentrations (10 mg/mL, 5 mg/mL and 2.5 mg/mL) using olive oil, mimicking commercial cannabis preparations.

Animal care and treatment

Thirty-five healthy juvenile male Wistar rats (Rattus norvegicus) were acquired post-weening from the Biomedical resource unit, University of Kwa-Zulu Natal, Durban, South-Africa. The rats (4 weeks of age, weighing between 80 and 100 g on arrival) were randomly divided into five groups, each of which containing seven animals. The rats were housed and maintained in standard polypropylene cages (350 × 350 × 220 mm) with a maximum of three rats per cage. The care and maintenance of the animals were in accordance to the requirements determined by the animal ethics committee of Nelson Mandela University (ethics number: A16-SCI-BCM-001). The housing facility was maintained at a constant room temperature of 22 °C with a 12-hour light cycle. Both food and water were available ad libitum.

Rats (for an 8 week period) were fed on a control diet of standard mice cubes (18% protein, 2.5% fat, 59% carbohydrate and 20.5% Other) (EPOL) and thereafter fed a high fat diet (26% protein, 35% fat, 26% carbohydrate and 13% other; Research diets, D12450K) for the remainder of the study. At 17 weeks, an intraperitoneal injection of streptozotocin (STZ) (Sigma) was performed on all groups at a treating concentration of 30 mg/kg rat mass, inducing an insulin impaired state. At 18 weeks, treatment was initiated and repeated every second day for all groups. The control group (STZ, n = 7) received an olive oil vehicle control at 500 µL/kg rat; three groups received cannabis resin dissolved in olive oil in increasing concentrations, treated at 500 µL/kg rat (1.25 mg/kg rat THC (STZ-CE1, n = 7); 2.5 mg/kg rat THC (STZ-CE2, n = 7) and 5 mg/kg rat THC (STZ-CE3, n = 7)); the final group was administered an aqueous metformin (Merck) solution orally at 50 mg/kg rat (STZ-MET) supplemented with an olive oil control at 500 µL/kg rat. Treatment ceased at 26 weeks of age.

Determining insulin resistance

Intraperitoneal glucose tolerance test (IPGTT) and fasting insulin concentrations were assayed to determine a diabetic state in accordance to the protocols established by Levendal et al., (2012) [13].

Animal euthanasia and measurements

Chemical euthanasia was administered intraperitoneally by pentobarbital injection at 40 mg/kg rat. Once euthanised, dissection commenced, and blood samples were collected directly from the heart by means of syringe extraction and stored in 0.11 sodium citrate (SMM chemicals). Blood plasma was obtained by means of centrifugation ( 1700 xg for 5 min; Eppendorf bench top mini spin) and stored at -20 °C [14]. Once the blood was drained, the prostate gland, seminal vesicles, testes and epididymides were removed and weighed.

Sperm counts and motility

A single epididymis was chosen and placed on a glass petri dish for vas deference sperm isolation. With a walking/squeezing motion utilising two forceps, vas deference sperm was ejected into 1 mL preheated (37 °C) sperm isolation media (0.2% (w/v) bovine serum albumin (Roche), 0.09% (w/v) glucose (Merck), 0.01% (w/v) sodium pyruvate (Gibco), 5.4 mmol/L KCl (Sigma), 4.2 mmol/L NaHCO₃ (Sigma), 1.3 mmol/L CaCl₂ (Sigma), 1.0 mmol/L MgSO₄ (Sigma), 0.44 mmol/L KH₂PO₄ (Sigma), 0.25 mmol/L Na₂HPO₄ (Sigma), 0.137 mol/L NaCl (Merck), buffered to pH 7.3) placed at the tip of the vas deferens. The sperm solution was then placed in a 1.5 mL Eppendorf and maintained at 37 °C for a 10-minute period. Sperm quantification and motility was analysed on the isolated sperm using a Neubauer chamber (Axiostar plus microscope, Zeiss). Percentage sperm motility was determined by counting a minimum of 100 sperm and classified into either motile or non-motile [15]. Sperm samples were stored in 10% glycerol at -20 °C for further analysis.

Serum testosterone assay

An enzyme-linked immunosorbent assay (Crystal Chem, Catalog #80,550) was used to determine the blood serum testosterone concentrations. The protocol was followed as per instructions and the resulting concentrations determined using an Epoch 2 microplate reader (Biotek).

Lipid peroxidation assay

The thiobarbituric acid assay was used to quantify the extent of lipid peroxidation in the vas deference sperm samples [16]. Sperm cells (4 × 10⁶) were centrifuged (4000 xg, Eppendorf 5804 R centrifuge) and resuspended in 1 mL phosphate buffered saline (PBS; 0.14 mol/L NaCl (Sigma), 8 mmol/L Na₂HPO₄ (Sigma), 3 mmol/L KCl (Sigma), 1 mmol/L NaH₂PO₄ (Sigma), buffered to pH 7.3). The centrifugation step was repeated once more. The resulting pellet was resuspended in 1 mL PBS followed by 3-minute sonication. The sperm solution was transferred to a glass test tube and 500 µL thiobarbituric acid reagent was added (23 mmol/L 2-thiobarbituric acid (BDH laboratory reagents), 62.5 mmol/L NaOH (Merck), 100 mL deionised H₂O and 100 mL glacial acetic acid (Merck)). The glass test tubes were covered in aluminium foil and placed in a boiling water bath for 60 min. The test tubes were cooled for 10 min at room temperature and the solutions centrifuged for 10 min at 4000 xg. From the supernatant, 100 µL was removed and analysed spectrophotometrically at 534 nm. Concentrations were determined according to a malondialdehyde concentration curve.

Enzyme assays

An indication of aerobic and anaerobic respiration rates were tested by determining the activities of lactated dehydrogenase (LDH, Anaerobic) and citrate synthase (CS, Aeorobic). Enzyme assays were performed on sperm samples which were sonicated for 3 min in sperm isolation media followed by centrifugation (700 xg; 10 min at 4 °C) using an Eppendorf 5804 R benchtop centrifuge.

The supernatant was assayed spectrophotometrically for LDH and CS activity as previously described [17, 18]. LDH Reaction buffer consisted of 94 mM Hepes (pH 7.0), 0.2 mM nicotinamide adenine dinucleotide (reduced form) (NADH) and 2 mM pyruvate, reaction as monitored at 340 nm for 10 min. CS reaction buffer consisted of 34 µM Acetyl-CoA, 95 mM Hepes (pH 8.0), 0.1% Triton X-100, 950 µM DTNB and 474 µM oxaloacetic acid and was monitored at 412 nm for 10 min.

Activity was expressed as product formed (µM) per protein (mg) per minute of reaction. Protein concentrations were quantified using the method of Bradford (1976) [19].

Statistical analysis

Analysis was performed on Microsoft Excel (version 1803) utilising a Real statistics resource pack, version 5.9. Results were determined to be significantly different (Pvalue less than 0.05) by means of student’s t-test assuming equal variance. All results are reported as average with error bars indicating standard error of mean unless stated otherwise.

Results

Insulin resistance

IPGTT analysis indicated a significant increase (P = 0.011) in fasting serum glucose levels (Fig. 1 A) between rats injected with streptotozin (STZ) and measurements taken when solely on a high fat diet (HFD). A significant decrease (P = 0.027) in fasting serum insulin concentration were observed after STZ treatment.

Fig. 1.

Fasting blood glucose (A) and insulin serum (B) concentrations of rats exposed to a high fat diet and STZ injection. Results indicate averages with error bars representing standard error (n = 7)

Organ masses

No significant difference was observed in the body, testes, epididymides, prostate gland and seminal vesicles of rats treated with cannabis or metformin (Table 1).

Table 1.

Average body, testis, epididymis, prostate and seminal vesicle mass in male rats exposed to a high fat diet, injected with streptozotocin and treated with various concentrations of cannabis extract and metformin (± SEM)

Mass (g)	Treatments
	STZ (n = 7)	STZ-CE1 (n = 7)	STZ-CE2 (n = 7)	STZ-CE3 (n = 7)	STZ-Met (n = 7)
Body	456.16 ± 8.94	494.74 ± 18.45	504.09 ± 19.53	455.06 ± 23.90	446.21 ± 16.51
Testis	1.69 ± 0.06	1.74 ± 0.04	1.77 ± 0.07	1.63 ± 0.03	1.68 ± 0.06
Epididymis	1.17 ± 0.06	1.26 ± 0.05	1.26 ± 0.05	1.06 ± 0.03	1.08 ± 0.04
Prostate gland	1.85 ± 0.15	1.91 ± 0.15	2.08 ± 0.19	1.98 ± 0.11	2.11 ± 0.20
Seminal vesicle	1.27 ± 0.19	1.22 ± 0.16	1.20 ± 0.18	1.27 ± 0.14	1.45 ± 0.09

Sperm counts and motility

No significant change in sperm count was observed for rats treated with cannabis. There was a significant (P = 0.035) increase in sperm count for the STZ-MET group when compared to the STZ control (Fig. 2). A concentration dependent decrease in sperm motility can be observed with an increasing concentration of cannabis treatment, resulting in a highly significant (P = 0.009) decrease in motility for the STZ-CE3 group when compared to the STZ control.

Fig. 2.

Sperm counts (A), and corresponding motility (B) of STZ injected rats treated with various concentrations of cannabis, and metformin. Results indicate averages with error bars representing standard error (n = 7)

Sperm lipid peroxidation

A highly significant (P = 0.009) decrease in lipid peroxidation was observed in the STZ-CE2 group when compared to the STZ control (Fig. 3). No significant changes were observed for the various cannabis concentrations tested or the metformin treatment.

Fig. 3.

The extent of lipid peroxidation (compared to malondialdehyde control) observed in vas deference sperm isolated from STZ injected rats exposed to various cannabis concentrations, and metformin treatment. Results indicate averages with error bars representing standard error (n = 7)

Serum testosterone concentrations

A general decrease in serum testosterone concentration is observed for all treated groups, however, this was not significant (Fig. 4). The STZ-CE3 and STZ-MET treated groups displayed a trend towards a significant decrease with P values of 0.11 and 0.14 respectively.

Fig. 4.

Serum testosterone concentrations of STZ injected rat exposed to various cannabis concentrations, and metformin. Results indicate averages with error bars representing standard error (n = 7)

Enzyme activities

LDH activities (Fig. 5 A) indicated an increasing trend in activity, with an increase in cannabis concentration, however, only at STZ-CE3 (5 mg/kg) a significant increase in activity (P = 0.065) was obtained as compared to the untreated STZ group. The STZ-MET group indicated a highly significant (P = 0.002) increase in LDH activities when compared to the STZ group. A significant increase in citrate synthase was only noted for the STZ-CE1 group.

Fig. 5.

LDH (A) and CS (B) activity of whole sperm isolated from the vas deference of rats injected with STZ and treated with various concentrations of cannabis, and metformin. Results indicate averages with error bars representing standard error (n = 7)

Discussion

This investigation aimed to determine if metformin or cannabis influences sperm and male reproductive organ parameters and how these treatments should be considered when a low fecundity is already prominent in diabetic males.

The development of a diabetic state was induced in rats fed on a high fat diet followed with an injection of streptozotocin. An insulin impaired state was confirmed with a significant increase (P = 0.011) in fasting glucose levels complimented with a significant decrease (P = 0.027) in fasting insulin concentrations (Fig. 1) [20, 21].

None of the treatments administered indicated a significant change in reproductive organ parameters (Table 1). In regard to sperm physiology, a concentration dependent decrease in sperm motility was observed with increasing concentrations of cannabis administered (Fig. 2) inducing a highly significant (P = 0.009) reduction in motility in diabetic rats treated at the highest concentration of cannabis (5 mg THC/ kg rat). Similarly, Hong et al. 1982 and Whan et al. 2006 determined that sperm motility decreased with an increasing concentration of THC in vitro for non-diabetic rats [22, 23]. The mechanism by which THC influences sperm motility is unknown, however, it is believed that the activation of cannabinoid receptors influence energy homeostasis sufficiently to inhibit the glycolysis dominated sperm migration [24]. Metformin increased the total vas deference sperm count significantly (P = 0.035) when compared to the STZ control. This increase in sperm count may be due to the ability of metformin increasing follicle stimulating hormone in testes and/or decreased oxidative stress in the testes [25].

The oxidative stress of the spermatozoa showed no significant change for the majority of the treatment groups excluding STZ-CE2. A highly significant (P = 0.009) decrease in lipid peroxidation (Fig. 3) was observed which may be dependent on the antioxidant properties of cannabinoids [26]. The same decrease in lipid peroxidation, however, was not observed at higher concentrations of cannabis treatment which can be linked to the biphasic and triphasic binding properties of cannabinoids to the specific receptors [27].

Although not significant, change in serum testosterone concentration levels were observed with both the treatment of cannabis and metformin (Fig. 4). Although this data was not significant, it should be noted that a more sensitive method such as mass spectrophotometry analysis may prove useful, where, similar decreases in testosterone have been reported previously in non-diabetic models and has only focussed on a single cannabinoid i.e. cannabidiol (CBD) [28, 29].

Only the lowest cannabis concentration (STZ-CE1) significantly influences citrate synthase activity indicating that the mitochondrial number or the rate of oxidative phosphorylation can only be altered by the lowest cannabis concentration (1.25 mg/kg) relative to the untreated control [30, 31]. There was, however, a highly significant increase in sperm LDH activity when treated with metformin and the highest cannabis concentration (STZ-CE3). This increase in LDH activity was also noted previous studies [29, 30, 31 and 32] for metformin and has in part been attributed to an increased sperm counts, which was also observed in this study for metformin, Fig. 1 [33].

To conclude, depending on the concentrations of the various cannabinoids or the ratio of these cannabinoids in the medicinal products used by individuals who are self-medicating, they may experience decreased testosterone concentrations and sperm motility. The extent of the adverse effects was concentration dependent and at a lower concentration of THC (1.25 mg/kg rat) limited negative results were observed. Metformin in comparison showed an increase in sperm counts and lactate dehydrogenase activity which may be beneficial in terms of sperm production and fertility comparable to literature [34–37]. It is important to note that this research was performed on a high fat diet induced rat model that was insulin resistant and therefore the effects observed may be different in humans. When fertility is of concern in young diabetic males, treatment with metformin may be preferred as the sole treatment to limit the negative effects observed.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contribution

All authors contributed in the acquisition of data, analysis, interpretation and manuscript preparation.

Funding

Funding provided by the National Research Foundation South Africa (bursary); Nelson Mandela University (operational expenses).

Data Availability

Available on request.

Code Availability

Not applicable.

Declarations

Conflict of interest

No known conflict of interest.

Ethics approval

The care and maintenance of the animals were in accordance to the requirements determined by the animal ethics committee of Nelson Mandela University (ethics number: A16-SCI-BCM-001).

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40200-022-01079-z.