Truong et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00468-3(2023) 24:6

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Sperm capacitation and transcripts levels
are altered by in vitro THC exposure
Vivien B. Truong1, Ola S. Davis1, Jade Gracey1, Michael S. Neal2, Jibran Y. Khokhar3 and Laura A. Favetta1*
Abstract
Background Delta-9-tetrahydrocannabinol (THC) is the primary phytocannabinoid responsible for the psychoactive
properties of cannabis and is known to interact with the endocannabinoid system, which is functionally present
in the male reproductive system. Since cannabis consumption is the highest among reproductive aged males, thecurrent study aimed to further investigate the effects of THC exposure to phenotypical, physiological, and molecularparameters in sperm. Bull sperm of known fertility were used as a translational model for human sperm and subjected
to in vitro treatment with physiologically relevant experimental doses of THC. Sperm parameters, capacitation,
apoptosis, and transcript levels were evaluated following treatment.
Results Motility, morphology, and viability of bovine sperm was unaltered from THC exposure. However, 0.32µM of
THC caused an increased proportion of capacitating sperm (p < 0.05) compared to control and vehicle group sperm.
Transcriptome analysis revealed that 39 genes were found to be differentially expressed by 0.032µM THC exposure,
196 genes were differentially expressed by 0.32µM THC exposure, and 33 genes were differentially expressed by
3.2µM THC. Secondary analysis reveals pathways involving development, nucleosomes, ribosomes and translation,and cellular metabolism to be significantly enriched.
Conclusion Phytocannabinoid exposure to sperm may adversely affect sperm function by stimulating premature
capacitation. These findings also show for the first time that spermatozoal transcripts may be altered by THC exposure.
These results add to previous research demonstrating the molecular effects of cannabinoids on sperm and warrant
further research into the effects of cannabis on male fertility.
Keywords Cannabis, THC, Sperm, Capacitation, Transcriptome, Fertility, Motility, Morphology, Apoptosis*Correspondence:
Laura A. Favetta
lfavetta@uoguelph.ca
1
Reproductive Health and Biotechnology Lab, Department of Biomedical
Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Rd.
East, N1G 2W1 Guelph, ON, Canada
2
ONE Fertility, Burlington, ON, Canada
3
Department of Anatomy and Cell Biology, Western University, London,
ON, Canada

Introduction
An estimated 50 million couples are affected by infertility globally, a population that continues to grow [1].
Male factor infertility is suggested to account for 20%
of instances of infertility and plays a role in another 30%
of cases [2]. Since natural biological causes of infertility
cannot account for all cases, environmental and lifestylevariables, including obesity, exposure to endocrine disrupting compounds, cigarette smoking, drug and alcohol
use have gained significant attention in fertility studies.
Cannabis is recognized to be the most widely consumed recreational substance globally, with over

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Truong et al. BMC Molecular and Cell Biology(2023) 24:6

6.4 million Canadians and 48.2 million Americans having reported cannabis use in 2019 alone [3, 4]. It is known
that men consume more cannabis compared to womenand are also more likely to consume higher potency
products [3, 5–7]. The overall high prevalence of cannabis use in recent years coincides with legislative changes
permitting therapeutic cannabis, such as Nabilone andDronabinol, which have been prescribed for anti-emetic
purposes [8]. Cannabinoids have also been used to alleviate spasticity in multiple sclerosis patients and have beenshown to have anti-anxiolytic, analgesic, and anti-epileptic properties [9, 10]. Medicinal cannabis use in the US
alone increased over four-fold between 2016 and 2020
based on medicinal cannabis program enrollment [11].
Continual support of legalization and increasing social
acceptability of cannabis use raise concerns about how
cannabis may impact fertility and reproductive health,especially among reproductive aged men [7].
Delta-9-tetrahydrocannabinol (THC) is the phytocannabinoid responsible for inducing the psychoactive
effects of cannabis, including euphoric feelings and cognitive impairment [12]. While delta-9-tetrahydrocannabinol is the primary psychoactive cannabinoid naturally
derived from cannabis sativa, several other isoforms of
THC have been identified including delta-8-tetrahydrocannabinol which contributes to approximately 10%
of THC content in cannabis [13, 14]. Other major isoforms of THC include delta-10-tetrahydrocannabinol
and delta-6a(10a)-tetrahydrocannabinol, which are synthetic isomers [15]. Furthermore, levels of THC foundin cannabis continues to trend upwards, due to selective breeding of the cannabis plant to cater to consumer
preference for higher potency products [16, 17]. The useof THC in the remaining text of this paper will refer todelta-9-tetrahydrocannabinol.
THC can activate cannabinoid receptors 1 and 2(CB1/2), belonging to the body’s endocannabinoid system (ECS) [18]. The ECS is a pro-homeostatic system ofreceptors, enzymes, and ligands coined as endocannabinoids (eCBs) [18, 19]. Anandamide (AEA) and 2-arachidonylglycerol (2-AG) are the two endogenously produced
cannabinoids, coined as endocannabinoids (eCBs),belonging to this system [18, 20]. The ECS was first
identified in the central nervous system, but has since
been confirmed to be present in the male and female
reproductive systems [21, 22]. Functional cannabinoid
receptors and eCBs present in mammalian sperm play asignificant role in modulating sperm motility, viability,
and capacitation [21, 23, 24]. In this regard, THC behaves
as an exocannabinoid, stimulating CB1 and CB2 as a partial agonist [25].
The working and dynamic relationship between the
ECS and sperm function has been successfully studied.
In humans, eCB levels in seminal fluid were found to

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be inversely associated with fertility [21]. These results
were corroborated by the work of Amoako et al. [26] who
reported reduced seminal AEA levels observed among
asthenozoospermic and oligoasthenoteratozoospermic
men. Furthermore, stimulation of either cannabinoid
receptors has been found to negatively impact sperm
motility and viability in human sperm and inhibit capacitation and the acrosome reaction in boar sperm [23,26–28].
Observational studies conducted among cannabis users also show detrimental effects of cannabis on
sperm parameters, sperm epigenetics, and molecular andbehavioural endpoints in offspring. Most notably, several
studies report lower sperm concentrations among cannabis smokers and some suggesting that THC specifically
can negatively impact spermatogenesis [29–32]. Cannabis users are also more likely to exhibit abnormal spermmorphology, a traditional indication of sperm quality
and fertility [33–36]. Altered methylation patterns havealso been observed in sperm from humans and rodents
exposed to cannabis [32, 37]. Several of these methylation
changes were also identified in the offspring of male mice
exposed to cannabis during the preconception period[38]. Paternal cannabis exposure in rodents has also been
associated with long-term neurological and behaviouraldifferences in offspring [39, 40].
Despite the number of existing studies regarding therole of the ECS in sperm and observational studies on
cannabis users, there have been limited explorations
on the direct effect of THC to sperm. Whan et al. [41]explored the effects of THC on fresh human sperm byusing concentrations of THC ranging from 0.032µM to
4.8µM, representing levels of THC found after therapeutic use of Marinol, an FDA approved cannabis product, and recreational cannabis use [42]. Both progressive
sperm motility and spontaneous acrosome reaction were
found to be inhibited by THC in a dose-dependent manner [41].
There have also been limited investigations on changes
to sperm transcripts in response to exogenous agents,
despite their abundance and evidence of differential gene
expression between fertile and infertile sperm [43–45].
In addition, an essential subset of early embryonic transcripts is known to be exclusively derived from sperm[46].
In this study we aimed to evaluate the effects of delta9-tetrahydrocannabinol (THC), the dominant naturally
occurring isomer, on sperm motility and morphology
as traditional indicators of sperm quality, in addition to
viability and capacitation, which reflect physiologicalcompetence [35, 47–49]. Lastly, we explored the potential
effects of THC on sperm transcriptome as sperm transcripts are reflective of fertility [43–45]. We hypothesizedthat THC would adversely affect motility, morphology,

Truong et al. BMC Molecular and Cell Biology(2023) 24:6

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Fig. 1 Motility characteristics following THC-treatment. Effect of therapeutic (low THC; 0.032 µM THC) and recreational (mid and high THC; 0.32 µM THC
and 3.2 µM THC, respectively) concentrations of THC on (A) percentage progressive motility, (B) percentage non-progressive motility, and (C) percentage
non-motile sperm. Motility was determined by manual sperm assessment of a minimum of 100 sperm per replicate after 6 and 12 h of exposure using a
Makler counting chamber. Error bars represent average ± SEM, n = 4

and viability, inhibit capacitation, and alter transcriptlevels. Owing to the constrains of human sperm use,the bovine model was chosen in the present study for
its morphological and physiological similarity to humansperm [50, 51].

Results
Effects of THC on motility and morphology

A total of four biological replicates were assessed for bothmotility and morphology following THC treatment for
6- and 12- hours, representing individual ejaculates fromfour bulls. These treatment lengths were determined byinitial motility and morphology experiments after treatment for various timepoints (Additional File 1, 2).

Sperm were categorized based on progressive motility, non-progressive motility, and non-motile. As shownin Fig. 1A-C, THC exposure for 6- or 12-hours at any
concentration did not significantly affect motility. THCtreated sperm did not have significantly altered spermmorphology when evaluated as total percentage of cumulative abnormal sperm assessed or as total percentage ofassessed sperm with head, neck, or tail defects (Fig. 2).
Baseline sperm characteristics of the bulls used in subsequent motility and morphology experiments can befound in Additional File 3.
Effects of THC on sperm viability

Sperm viability was measured from a total of four biological replicates following THC exposure for 6- andTruong et al. BMC Molecular and Cell Biology(2023) 24:6

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Fig. 2 Morphology characteristics following THC-treatment. Effect of therapeutic (low THC; 0.032 µM THC) and recreational (mid and high THC; 0.32 µM
THC and 3.2 µM THC, respectively) concentrations of THC on (A) overall sperm morphology, (B) head defects, (C) neck defects, and (D) tail defects. Morphology was manually determined using a minimum of 200 sperm per group per replicate after 6 and 12 h of treatment. Error bars represent ± SEM, n = 412-hours, representing sperm from individual ejaculates
from four bulls. Flow cytometry density plots indicateddifferent quadrants for sperm subpopulations in eachtreatment group based on Annexin/PI staining patterns(Fig. 3A-F). In the 6-hour study, DTT-treated group
had significantly less live sperm and more late apoptotic
sperm compared to the control and vehicle (p < 0.05),

whereas THC treatment did not affect viability (Fig. 3G).
Similarly, in the 12-hour study, DTT induced early andlate apoptosis (p < 0.05) but THC treatment did not alter
viability (Fig. 3H).

Truong et al. BMC Molecular and Cell Biology(2023) 24:6

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Fig. 3 Sperm viability characteristics following THC-treatment. Effects of therapeutic (low THC) and recreational (mid and high THC) concentrations of
THC on viability. Representative density plots of sperm in the (A) Dithiothreitriol (DTT) or apoptosis positive control group, (B) control group, (C) vehicle
group (0.01% ethanol), (D) low THC group (0.032 µM THC), (E) mid THC group (0.32 µM THC), and (F) high THC group (3.2 µM THC). In each plot, the bottom
left quadrant (PI and FITC negative) represent live sperm, the top left quadrant (PI positive, FITC negative) represent necrotic sperm, the top right quadrant(PI positive, FITC positive) represent late apoptotic sperm, and bottom right quadrant (PI negative, FITC positive) represent early apoptotic sperm. Average
percentage of live, early apoptotic, late apoptotic, and necrotic proportions of sperm following (G) 6- and (H) 12-hours of treatment. PI, propidium iodide;FITC, Annexin V-FITC. Error bars represent ± SEM, n = 4. Asterisk (*) indicate p < 0.05

Truong et al. BMC Molecular and Cell Biology(2023) 24:6

Effect of THC on sperm capacitation

Sperm capacitation was measured from a total of fourbiological replicates following 6-hours of treatment,
representing individual ejaculates from four bulls. Flowcytometry density plots showed the distribution of the
sperm populations after incubation with M540/YP1(Fig. 4A-F). Live/Non-capacitating and apoptotic proportions of sperm were unchanged by THC treatment(Fig. 4G-H), while the Mid-THC treated group exhibited significantly higher proportions of live-capacitating
sperm in comparison to the control and vehicle (p < 0.05)(Fig. 4I). Heparin treatment also caused increased capacitation (p < 0.05) (Fig. 4I).
Effect of THC on transcriptome

To determine THC effects at the gene expression level,a transcriptome analysis was performed on two biological replicates of sperm. The first biological replicate
consisted of RNA from sperm of a single bull, while thesecond biological replicate consisted of RNA from apooled sample of sperm originating from three separatebulls (to mitigate individual variation). Differential gene
expression analysis revealed 100 DEGs between spermof the control and vehicle groups, 77 DEGs between
the vehicle and low-THC, 321 between the vehicle and
Mid-THC, and 86 between the vehicle and high-THC(Fig. 5A). After excluding unknown genes and genesthat were differentially expressed between the control
and vehicle groups, 39, 196, and 33 DEGs remained
between the vehicle and low-THC, mid-THC, and highTHC sperm, respectively (Fig. 5B). There were 7 overlapping DEGs between the Low- and Mid-THC sperm, 14
overlapping between the Mid- and High-THC sperm,
and four overlapping between the Low- and High-THC
sperm. Three transcripts were detected at lower levels
in THC-treated sperm compared to the vehicle-sperm,
regardless of THC concentration; ANKRD31 (Ankyrin
repeat domain 31), B9D1 (B9 domain 1), and ROPN1L(rhophilin associated tail protein 1-like). Full DEG lists
can be found in Supplementary Tables 1–3 in Additional
File 4.
Secondary analysis of the DEG lists using DAVID 6.8
revealed several enriched gene ontology (GO) groups.
Among the DEGs from the low-THC treatment sperm,
GO terms relating to nucleosome assembly, ribosomes,
oxidative phosphorylation, in utero development, andmitochondrial small ribosomal subunit were enriched(Fig. 5C). Among DEGs from the Mid-THC treatedsperm, GO terms relating to the nucleosome, multicellular organism development, ribosomes, glycolysis/gluconeogenesis, and the cell cycle were enriched (Fig. 5D).
Finally, GO terms ribosome/translation and ubiquitinlike protein conjugation were enriched based on the DEG
list of High-THC treated sperm (Fig. 5E).

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Discussion
Sperm function can be influenced by exogenous compounds present in the body through several mechanisms.
Previous literature has associated THC use in reproductive aged men with increased spermatid apoptosis, lowsperm count, and teratozoospermia [29–34]. The presence of the ECS in the male reproductive tissues and its
significant contributions to sperm activity warrant additional investigation into the potential effects of exocannabinoids, including the psychoactive phytocannabinoid
THC, on sperm function using an in vitro model.
Regulated levels of eCBs are necessary for the development of sperm as well as mature sperm competence [21,52, 53]. An existing gradient of eCBs decreasing fromseminal plasma to oviductal and follicular fluid is suggested to spatio-temporally regulate sperm motility andcapacitation by maintaining a suppressive effect on sperm
until they are in closer proximity to the oviduct [54, 55].
Furthermore, stimulation of the cannabinoid receptors
has generally been found to be inhibitory towards sperm
activity [23, 24, 28].
In the present study, we studied the effects of physiologically relevant concentrations of THC (0.032µM,
0.32µM, and 3.2µM) representative of plasma levels of
THC following therapeutic (low dose) and recreational(mid and high dose) cannabis use [41, 42, 56]. It should
be noted that due to high lipophilicity, amount of THC in
fatty tissues, such as the epididymis, following repeated
THC exposure accumulates at levels up to 80 times
higher than amounts in the brain and plasma [57]. As aresult, the half-life of THC ranges from 6 min to as longas 22 h [58]. To our knowledge, there has only been one
research group to quantify THC levels in the semen of
cannabis users, who measured THC in semen 8–10 hfollowing cannabis consumption and reported THC concentrations ranging from 0.87 to 0.97 ng/mL (equivalent
to 2.7–3.1nM), below the range of concentrations usedin our study [59]. However, since these findings are not
likely to represent peak seminal fluid THC levels occurring sooner after consumption, we believe that the use ofpeak plasma concentration to approximate THC exposure was more appropriate.
Here, we assessed motility and morphology as parameters that have been traditionally used as indicators ofsperm quality [35, 47]. We found no significant effects of
THC on sperm motility or morphology, contrary to our
initial hypothesis and previous literature documenting
detrimental effects [23, 31, 41, 60]. It is likely that the lackof morphological changes after THC exposure in thisstudy are a result of the use of mature sperm, whereas
previous observations of teratozoospermia among cannabis users reflects the impact of THC during spermatogenesis. Interestingly, biphasic responses to THC in sperm
have been recognized, where 2.5nM of methanandamideTruong et al. BMC Molecular and Cell Biology(2023) 24:6

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Fig. 4 Sperm capacitation status following THC-treatment. Effects of therapeutic (low THC) and recreational (mid and high THC) concentrations of
THC on capacitation. Representative density plots of sperm in the (A) Heparin or capacitation positive control group, (B) control group, (C) vehicle
group (0.01% ethanol), (D) low THC group (0.032 µM THC), (E) mid THC group (0.32 µM THC), and (F) high THC group (3.2 µM THC). In each plot, the bottom left quadrant (Yo-Pro-1 and Merocyanine-540 negative) represent live and non-capacitating sperm, the top left quadrant (Yo-Pro-1 negative andMerocyanine-540 positive) represent live and capacitating sperm, and the two right quadrants (Yo-Pro-1 positive) represent apoptotic sperm. Average
percentages of (G) live non-capacitating sperm, (H) apoptotic sperm, and (I) live capacitating sperm. YP1, Yo-Pro-1; M540, Merocyanine-540. Error bars
represent ± SEM, n = 4. Asterisk (*) indicate p < 0.05

Truong et al. BMC Molecular and Cell Biology(2023) 24:6

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Fig. 5 THC-induced changes to spermatozoal transcripts. Differentially expressed genes (DEGs) and gene ontology (GO) terms in sperm treated withtherapeutic (low THC; 0.032 µM) and recreational (mid and high THC; 0.32 µM and 3.2 µM, respectively) concentrations of THC. (A) Total number of DEGs
(upregulated and downregulated), (B) Filtered DEG to exclude unknown genes and DEG of the vehicle group, and enriched GO terms in the (C) low-THC
DEGs, (D) mid-THC DEGs, and (E) high-THC DEGs. Dark and light boxes indicate decreased and increased expression, respectively, from THC treatment

Truong et al. BMC Molecular and Cell Biology(2023) 24:6

(Met-AEA), a stable analog of AEA, inhibited motility,
but 0.25nM of met-AEA stimulated motility, suggestingthat exocannabinoid effects on sperm are concentrationdependent [61].
Although the exact mechanisms are not fully understood, spermatozoal apoptosis involves the release ofcytochrome C, caspase activation, and phosphatidylserine (PS) externalization, similar to somatic cell apoptosis[62–64]. Interestingly, the unique compartmentalizationof sperm limit the availability cytoplasmic enzymes,
which reduce the ability of the sperm to combat oxidative stressors [65]. Stimulation of the ECS is generally
regarded to be pro-apoptotic as cannabinoid receptor
stimulation causes ceramide accumulation leading tocellular stress responses and activation of the mitochondrial apoptotic pathway [66, 67]. Here, we assessed theeffects of THC on sperm viability as a reflection of sperm
physiology and potential ECS stimulation. We foundno changes to viability resulting from THC exposure,in contrast to our hypothesis. Interestingly, Rossato etal. [23] report that 10µM of AEA, but not 0.1 or 1.0µM,
eliminated sperm viability in vitro. Previous reports
also implicate levels of THC beyond 30µM THC with
the inhibition of mitochondrial membrane potential insperm, a common indicator of apoptosis [68]. As a result,
it is possible that apoptosis is only induced at concentrations substantially higher than the ones used in the present study.
The results of our motility, morphology, and apoptosis
experiments may be explained by considering our useof cryopreserved bull sperm as an experimental model.
Both extensive washing before and after the cryopreservation and rigorous selection process of bull sperm during commercial sperm cryopreservation leaves only themost robust sperm, which are reported to be more resistant to the effects of exocannabinoids [41]. On the other
hand, cryopreservation necessitates removal of roundsomatic cells and seminal plasma, components which
would otherwise be present during THC-sperm interactions and provide possible protective effects [68]. Lastly,
the cryopreservation process is known to induce oxidative damage and apoptosis in sperm, which may have
limited our potential to observe the effects of THC toviability [69, 70].
To further investigate THC-induced changes in sperm
physiology, we examined capacitation, a necessary seriesof cellular and biochemical changes which allow sperm
to propel through the female reproductive tract and prepare to fertilize the ovum [48, 71]. We observed that 6 hof 0.32µM of THC exposure, our mid-THC concentration, significantly increased the proportion of capacitating sperm compared to the sperm in the vehicle group,
based on positive M540 and negative YP1 staining. Interstingly, sperm in the same group did not exhibit altered

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motility, which is typically observed in capacitating
sperm through hypermotility. Previous research indicates
conflicting evidence regarding the relationship between
cannabinoids and capacitation. AEA and THC have beenshown to inhibit the acrosome reaction, which is widely
accepted as the culminating event of capacitation and isnecessary for sperm fertilization competence [23, 41, 61].
On the other hand, 2-AG has been observed to increase
the acrosome reaction in human sperm and met-AEA
has been found to induce bull sperm capacitation [24,72]. When intracellular calcium was measured as an indicator of capacitation, Gervasi and collegues [72] report
only the two median concentrations of met-AEA eliciteda significant change, similarly to the results we observedin the present study.
Our results indicate that THC can impact sperm physiology by promoting membrane disorder characteristic of
capacitation. Although capacitation is a crucial aspect ofsperm function, premature capacitation may unfavourably affect fertility by stimulating early hypermotility
leading to exhaustion of sperm ATP, inability to reach theovum, and premature death [54]. Moreover, prematureacrosome reaction also impairs subsequent fertilization[48].
To our knowledge, this is the first study to explore andreport effects of THC to transcripts in mature sperm.
Cannabinoid receptor signalling in somatic cells canlead to downstream changes in gene expression [73–75].
However, a lack of transcriptional machinery limits transcriptional activity in sperm [76, 77]. Nonetheless, transcripts in mature sperm are known to be altered fromcryopreservation and exposure to endocrine disrupting
compounds, suggesting a mechanism for altered transcript levels [78–80].
We report over 200 DEGs in sperm exposed to THC,
many relating to development, cellular metabolism, ribosomes and translation, and the nucleosome. It is noteworthy that the secondary gene ontology study performed
found enrichment of DEGs relating to in utero development in the low-THC group, where all developmentally
related genes had decreased expression, and multicellular organism development mid-THC group, where thesegenes had increased expression. We also report that all
THC-treated sperm, regardless of concentration, sharedenrichment in genes relating to ribosomes and translation, largely as a result from the differential expression of
mitochondrial ribosomal proteins. Although sperm are
generally thought to be translationally silent due to a lackof cytoplasmic ribosomes, Gur & Breitbart [81] described
the presence of protein synthesized by mitochondrial
ribosomes in mature sperm. Further research suggeststhat specific transcripts in mature sperm are activelytranslated by mitochondrial ribosomes during capacitation and the inhibition of mitochondrial translation isTruong et al. BMC Molecular and Cell Biology(2023) 24:6

detrimental to capacitation, motility, and fertilization [81,82]. These findings might provide insight on the considerable number of genes found to be decreased in spermof the mid-THC group, which we also reported to have asignificantly increased proportion of capacitating sperm.
Since it is unlikely that changes in transcript abundance
are due to transcriptional activity for reasons described
previously, we speculate that the decrease in transcripts
in this treatment group specifically was a result of transcript turnover stemming from increased mitochondrial
ribosome activity during capacitation. Moreover, it ispossible that the wide variety of transcriptomic changes
observed in the presented study is a result of either translation dependent transcript turnover, accumulation, or
altered stability. We can even speculate that, given thelimited research on the mechanisms of transcript turnover in sperm, transcriptomic changes may be due to theinhibition of transcript degradation or decreased stability, both through mechanisms that have not yet been
investigated in sperm. This is a limitation of this study,
that warrants further experiments to confirm specifictranscripts changes and to clarify the reason of thesetranscriptional changes.
In conclusion, our findings that THC induces capacitation provide evidence to support the growing body offacts suggesting that cannabinoids impact sperm physiology. To our knowledge, this is the first study to investigate the impacts of cannabinoids on transcripts inmature sperm. We report that THC can induce changes
to sperm transcript levels, although additional validation
studies should be performed prior to making functional
conclusions. This study also utilized physiologically relevant concentrations of THC to mimic natural physiological responses as closely as possible in an in vitro
model. Altogether, these findings justify further researchon the cellular and molecular impacts of cannabinoids onsperm. In combination with previous work highlighting
the detrimental effects of THC on sperm development
and function, these results contribute to the growing
body of research seeking to address the implications ofcannabis use among reproductive aged men, the highest
consumers of cannabis.

Materials & methods
Reagents

All media and chemicals were purchased from Sigma
Aldrich (Oakville, ON, Canada) unless otherwise specified. THC stock was purchased as Dronabinol (Δ9-THC)
from Toronto Research Chemicals (Toronto, ON,
Canada).
Semen preparation & treatment

Cryopreserved bull semen from bulls with known fertility were acquired from Semex (Guelph, ON, Canada).

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For each bull, 200uL of cryopreserved semen containing approximately 50 million sperm was thawed andwashed using a discontinuous Percoll gradient describedby Nguyen et al. [80]. The washed sperm was then split
into one of five treatment groups containing 1mL of
media each; HEPES/Sperm TALP supplemented with
0.3% bovine serum albumin (Control), 0.01% of ethanol
diluted in the control group media (Vehicle), 3.2µM of
THC diluted in the control group media (High-THC),
0.32 µM of THC made by 10-times serial dilution ofHigh-THC media (Mid-THC), and 0.032µM of THC
made by 10-times serial dilution of Mid-THC media(Low-THC). THC concentrations used in this study weremeant to reflect plasma concentrations of THC following
therapeutic and recreational cannabis use as described by
Misner et al. [56]. Sperm were incubated at 38.5 °C with
5% CO2 until assessment.
Effects of THC on motility and morphology

Sperm were retrieved after incubation for 6 and 12 h in
the aforementioned conditions and centrifuged for 7 min
at 2,500 RPM. Supernatant was removed and sperm pellet was resuspended in 50uL of warmed HEPES/Sperm
TALP. For morphological assessment, 10uL of the resulting sperm suspension was spread onto a warmed glass
slide using the pipette method [83] and air dried before
being fixed overnight in a 3:1 methanol:acetic acid solution. Slides were then washed with water, stained witha Giemsa staining solution for 15 min, washed again,and evaluated under a Leica DMIRB microscope (Leica
Microsystems; Richmond Hill, ON, Canada). Morphology of a minimum of 200 sperm per treatment group per
biological replicate was manually assessed as previously
described by Nguyen et al. [80].
For motility assessments, 10uL of sperm from resuspended pellet was placed onto a prewarmed Makler
counting chamber and 90 s video recordings (25 frames
per second) containing a minimum of 6 fields generatedusing the EPView camera and software (Olympus Life
Sciences; Richmond Hill, ON, Canada). Recordings wereused to manually evaluate the motility of a minimum of
100 sperm per treatment group per biological replicate as
described by Nguyen et al. [80]. Both motility and morphology assessments were conducted by a single user andunder blinded conditions.
Effects of THC on sperm apoptosis and capacitation

Apoptosis was evaluated using the Annexin V-FITC
Apoptosis Staining/Detection Kit consisting of binding buffer, propidium iodide (PI), and annexin V-FITC(FITC) (Abcam, Cambridge, MA, USA). Sperm weretreated for 6 and 12 h and washed as previously described
before being resuspended in 180uL of warmed binding buffer. A sixth treatment group containing 0.7µM ofTruong et al. BMC Molecular and Cell Biology(2023) 24:6

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dithiotretriol (DTT) added 45 min before the end of thetreatment period was used as a positive control for apoptosis. Three additional groups consisting of no stain, only
FITC, and only PI controls required for flow cytometrycompensation were included. In a dark room, 5uL of PI
and FITC were added to each sample and incubated for
15 min before being assessed using the BD C6 Accuri
Flow Cytometer (BD Biosciences, Mississauga, ON,
Canada). FITC labels phosphatidylserine residues whichaccumulate on the outer leaflet of the lipid bilayer under
apoptotic conditions while PI, a DNA dye, enters the cellupon membrane compromise during necrosis [84, 85].
FITC negative/PI negative staining represents the live
sperm population, FITC positive/PI negative staining
indicates early apoptosis, FITC positive/PI positive indicates late apoptosis, and FITC negative/PI positive indicates necrosis [86].
To assess capacitation, sperm were incubated for
6 h before being washed and resuspended in 180uL ofwarmed HEPES/Sperm TALP. Merocyanine-540 (M540)
and Yo-Pro-1 (YP1) diluted in DMSO were added tosperm suspensions to achieve final staining concentrations of 50nM and 1.5µM, respectively. An additionalgroup containing 10uL of 2U/mL heparin, a glycoaminoglycan known to induce capacitation in bovine sperm,
was used as a positive control group [87, 88]. Three additional groups consisting of no stain, only M540, and only
YP1 controls required for flow cytometry compensation
were included. Samples were incubated in the dark before
being assessed by flow cytometry. M540 intercalates intocell membranes with increasing membrane disorderwhile YP1, a DNA binding dye, enters early apoptotic
cells with permeable membranes [89, 90]. Events that are
YP1 positive indicate an apoptotic sperm, M540 positive/YP1 negative events represent live and capacitating
sperm, and M540 negative/YP1 negative events represent
the live and non-capacitating sperm [91].
Flow cytometry from both apoptosis and capacitation
experiments were analysed using FlowJo v10 (BD Biosciences) using a minimum of 25,000 events per sample
after gating to remove debris. Quadrants were determined based on no stain controls.

Whitby, ON, Canada) at the David Braley Research Institute in Hamilton, Ontario, Canada. One biological replicate is representative of a single bull, while the second
biological replicate consists of sperm pooled from threedifferent bulls to mitigate individual variability.
Results of the transcriptome analysis were evaluatedusing Transcriptome Analysis Console (Thermo Fisher
Scientific). Differentially expressed genes (DEG) wereconsidered to be statistically significance if the foldchange was greater than ± 2 and had a p-value of less than
0.05. The Database for Annotation, Visualization andIntegrated Discovery (DAVID 6.8) was used to perform asecondary gene ontology analysis.

Transcriptome analysis

Funding
This project is supported by the General Purpose Account (# 072049) of LAF
through the Department of Biomedical Sciences, University of Guelph and theCanadian Graduate Scholarship (Master’s, NSERC) of VBT.

Washed sperm from 400uL of cryopreserved semen wereplaced in 4mL of each of the treatment groups described
previously for 6 h. Treated sperm pellets from two biological replicates were snap frozen and stored at -80 °C
until total RNA extraction, using the Qiagen miRNeasy
Micro RNA extraction kit according to manufacturer’s
instructions (Qiagen, Toronto, ON, Canada). RNA samples from two biological replicates of bull sperm underwent a transcriptome analysis using a GeneChip Bovine
Gene 1.0 ST Microarray (Thermo Fisher Scientific;

Statistics

Statistical analysis was performed on GraphPad Prism 6
using data from at least 3 biological replicates. All data
sets were subjected to the Kolmogorov-Smirnov test of
normality. Normally distributed data sets were analyzed
using one-way analysis of variance (ANOVA) and nonnormally distributed data sets were analyzed using the
Kruskal–Wallis test. Tukey’s post-hoc test was used todetermine statistical significance between the treatmentgroups.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-023-00468-3.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
Acknowledgements
The authors would like to thank Monica Antenos, Elizabeth St. John, Allison
MacKay, and all members of the Reproductive Health and Biotechnology Lab
in the Department of Biomedical Sciences at the University of Guelph for theirtechnical assistance and resourceful scientific discussion.
Authors’ Contribution
VBT wrote the main manuscript and performed all data analysis. VBT and OSD
carried out motility and morphology assessments. JG performed capacitation
experiments. VBT, LAF, MSN, and JYK conceptualized experiments. Finalmanuscript was read and approved by all authors.

Data Availability
The datasets used and/or analysed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Competing Interests
The authors declare no competing interests.

Truong et al. BMC Molecular and Cell Biology(2023) 24:6

Received: 20 November 2022 / Accepted: 22 February 2023

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