Testicles – spermatogenesis
Animal studies
ECS  involvement
In rat testes, AEA and 2AG are present as well as CB1 and CB2. CB1 and AEA appear involved in spermatogonia stem cell activity and spermatid formation while CB2 and 2AG appear involved in mitotic and meiotic stages. In addition, CB1 is found in fetal gonocytes (Migliaccio et al., 2018).
In bulls it was found that CB1 expression in spermatozoa was positively and significantly correlated to bull fertility (Kumar et al., 2018).
In mice it was shown that CB1 modulates/stabilizes sperm chromatin condensation during epididymal transit (as part of the sperm maturation process)(Chioccarelli et al., 2020).
2AG affects the in vitro functionality of human sperm by reducing motility, inhibiting capacitation and triggering the acrosome reaction (Francou et al., 2017).
Exposure of male mice to JWH-133, a selective CB2 agonist, decreased sperm count, impaired placental development and reduced offspring growth. These defects were associated with altered DNA methylation/hydroxymethylation levels at imprinted genes in sperm and conserved in placenta (Innocenzi et al., 2019).
In water buffalo, 1 or 10 μM of AEA reduced cryo-capacitation during cryopreservation and improves post-thaw sperm quality (Kumar et al., 2017a).
In another experiment, 1 nM AEA decreased sperm binding to oviduct explants from water buffalo. This effect was CB1-dependent and was not observed with 1 μM AEA (Kumar et al., 2017b), possibly indicating a bi-phasic effect of AEA.

Plant cannabinoids
In rats, Benin hemp extract (oral application of 2 or 10 mg/kg, with a small amount of CBN but no THC or CBC) increased sperm count and viability but not motility compared to extract from Nigerian hemp extract (high in THC)(Alagbonsi et al., 2019). This suggests that cannabinoid composition of extracts can influence sperm quality.
In rat semen treated with 1 mM THC sperm motility was reduced. This motility reduction was partially reversed by CB1 or CB2 inhibition and completely reversed by CB1 and CB2 inhibition(Alagbonsi and Olayaki, 2018). The spermatoxic effect of THC was ameliorated by melatonin. Please note that the THC concentration in this experiment was very high for human standards.
Interestingly, the same authors found melatonin exacerbated cannabis-induced gonadotoxicity in vivo (Alagbonsi et al., 2016)!
In male mice treated with 10 mg/kg THC for 30 days, no effect on CB1 expression or methylation, testicular or epididymal weight or histology or sperm concentration or motility was found, contradicting the notion that THC negatively impacts male fertility (López-Cardona et al., 2018).
In mice the effect of chronic oral CBD (15 or 30 mg/kg for 34 consecutive days) on the male reproductive system was tested. 15 mg/kg reduced the number of mounts and ejaculations while 30 mg/kg reduced fertility by 30% and litter number by 23% (Carvalho et al., 2018a). CBD treatment increased stages 1-6 and decreased stages 7, 8 and 12 of spermatogenesis, reduced epididymal spermatozoa by 38% and altered sperm morphology (Carvalho et al., 2018b). This suggests a negative effect of chronic CBD on male reproduction but please note that the dose is very high for human standards.
A review of the effects of CBD on the male reproductive system concludes that exposure to CBD is associated with a reduction in mammalian testis size, the number of germ and Sertoli cells in spermatogenesis, fertilization rates, and plasma concentrations of hypothalamic, pituitary and gonadal hormones. Moreover, chronic doses of CBD have impaired sexual behavior in mice (Carvalho et al., 2019).

Human studies
ECS involvement
Human testes contain 2AG, DAGL, NAPE-PLD, MAGL, ABDH2, FAAH and CB1 and CB2 suggesting a role for the endocannabinoid system in testicular physiology (Nielsen et al., 2019).
In both fertile and non-fertile men, CB1 and CB2 are expressed in sperm. This expression increases during sperm maturation but is significantly more upregulated in fertile men (Hazem et al., 2020).
Human sperm capacitation (by fibronectin) is under control of FAAH (reduced activity after 1 minute of capacitation, increased activity after 60 minutes) and is inhibited by CB1 or TRPV1 antagonists (Martínez-León et al., 2018) suggesting the endocannabinoid system controls the final stages of sperm maturation.
TRPV2 is expressed in human testicular peritubular cells (HTPCs). In cultured HTPCs, CBD was found to induce pro-inflammatory and angiogenic factors suggesting a role in (patho-) physiology (Eubler et al., 2018).
In human sperm ABDH2 is highly expressed in spermatozoa. Upon progesterone binding ABDH2 hydrolyses and depletes 2AG from the plasma membrane leading to sperm activation (Miller et al., 2016).
In human seminal vesicles CB1, CB2, GPR55, FAAH1 and FAAH2 are highly expressed. CB1, CB2 and GPR55 located to the pseudo-stratified columnar epithelium and varicose nerves (also characterized by the expression of vasoactive intestinal polypeptide and calcitonin gene-related peptide). Cytosolic staining for FAAH1 and FAAH2 was seen in cuboidal cells of all layers of the epithelium. No immunoreactivity was detected in the smooth musculature or nerve fibers (Ückert et al., 2017).

Plant cannabinoids
In human sperm exposed to 0.032, 0.32 or 4.8 M THC sperm motility and acrosome reactions were increasingly impaired with increasing THC concentrations (Whan et al., 2006). This impairment is relatively mild in the strong (90%) sperm fraction but more pronounced in weaker sperm (45% fraction). While this suggests a negative effect of THC on male fertility, this could also be interpreted as increased selection for stronger sperm.
Human sperm from cannabis users has an approximately 10% difference in DNA methylation pattern and a lower concentration of spermatids (Murphy et al., 2018).
A longitudinal study among sub-fertile men showed that men smoking cannabis (current or past use) had significantly higher sperm counts than men who had never smoked cannabis. Also men (with a history of) smoking cannabis were less than half as likely fall below WHO reference values for fertility (Nassan et al., 2019).
In a cohort of 200 Swiss men it was found that seminal fluid AEA levels and serum OEA levels were inversely correlated with sperm motility while semen PEA was positively linked to sperm concentration. Moreover, OEA and PEA in seminal fluid were associated with better sperm morphology. Interestingly, the concentrations of the same endocannabinoids measured in both blood and semen were not correlated, and the presence of THC metabolites in some individuals was linked to lower concentrations of endocannabinoids (Zufferey et al., 2020).
In the sperm of some, but not all, chronic cannabis users THC can be detected, demonstrating that THC can cross the blood-testicular-barrier. However, semen THC levels were only moderately correlated with serum THC and not correlated with urinary cannabinoid levels or sperm concentration/motility/morphology (Lee et al., 2020).

Literature:
Alagbonsi, I.A., and Olayaki, L.A. (2018). Melatonin attenuates Δ9-tetrahydrocannabinol-induced reduction in rat sperm motility and kinematics in-vitro. Reprod. Toxicol. Elmsford N.
Alagbonsi, A.I., Olayaki, L.A., Abdulrahim, H.A., Adetona, T.S., and Akinyemi, G.T. (2019). cannabinoid-deficient Benin republic hemp (Cannabis sativa L.) improves semen parameters by reducing prolactin and enhancing anti-oxidant status. BMC Complement. Altern. Med. 19, 132.
Alagbonsi, I.A., Olayaki, L.A., and Salman, T.M. (2016). Melatonin and vitamin C exacerbate Cannabis sativa-induced testicular damage when administered separately but ameliorate it when combined in rats. J. Basic Clin. Physiol. Pharmacol. 27, 277–287.
Carvalho, R.K., Souza, M.R., Santos, M.L., Guimarães, F.S., Pobbe, R.L.H., Andersen, M.L., and Mazaro-Costa, R. (2018a). Chronic cannabidiol exposure promotes functional impairment in sexual behavior and fertility of male mice. Reprod. Toxicol. Elmsford N.
Carvalho, R.K., Santos, M.L., Souza, M.R., Rocha, T.L., Guimarães, F.S., Anselmo-Franci, J.A., and Mazaro-Costa, R. (2018b). Chronic exposure to cannabidiol induces reproductive toxicity in male Swiss mice. J. Appl. Toxicol. JAT.
Carvalho, R.K., Andersen, M.L., and Mazaro-Costa, R. (2019). The effects of cannabidiol on male reproductive system: A literature review. J. Appl. Toxicol. JAT.
Chioccarelli, T., Manfrevola, F., Porreca, V., Fasano, S., Altucci, L., Pierantoni, R., and Cobellis, G. (2020). The cannabinoid Receptor CB1 Stabilizes Sperm Chromatin Condensation Status During Epididymal Transit by Promoting Disulphide Bond Formation. Int. J. Mol. Sci. 21.
Eubler, K., Herrmann, C., Tiefenbacher, A., Köhn, F.-M., Schwarzer, J.U., Kunz, L., and Mayerhofer, A. (2018). Ca2+ Signaling and IL-8 Secretion in Human Testicular Peritubular Cells Involve the Cation Channel TRPV2. Int. J. Mol. Sci. 19.
Francou, M.M., Girlea, J.L., De Juan, A., Ten, J., Bernabeu, R., and De Juan, J. (2017). Human sperm motility, capacitation and acrosome reaction are impaired by 2-arachidonoylglycerol endocannabinoid. Histol. Histopathol. 11911.
Hazem, N.M., Zalata, A., Elghobary, M., Comhaire, F., and Elabbasy, L.M. (2020). Evaluation of cannabinoid receptors Type 1 and Type 2 mRNA expression in mature versus immature spermatozoa from fertile and infertile males. Andrologia e13532.
Innocenzi, E., De Domenico, E., Ciccarone, F., Zampieri, M., Rossi, G., Cicconi, R., Bernardini, R., Mattei, M., and Grimaldi, P. (2019). Paternal activation of CB2 cannabinoid receptor impairs placental and embryonic growth via an epigenetic mechanism. Sci. Rep. 9, 17034.
Kumar, P., Mohanty, T.K., Kumaresan, A., Nag, P., Saraf, K.K., Kumar, V., Lathika, S., Nayak, S., and Bhakat, M. (2017a). Incubation of spermatozoa with Anandamide prior to cryopreservation reduces cryocapacitation and improves post-thaw sperm quality in the water buffalo (Bubalus bubalis). Anim. Reprod. Sci.
Kumar, V., Kumaresan, A., Kumar D S, P., Lathika, S., Nayak, S., Kishor Saraf, K., Nag B S, P., Chhillar, S., Kumar Datta, T., and Kumar Mohanty, T. (2017b). Anandamide exerts a suppressive effect on sperm binding to oviduct explants through CB1 receptors in the water buffalo (Bubalus bubalis). Anim. Reprod. Sci.
Kumar, V., Kumaresan, A., Nag, P., Kumar, P., Datta, T.K., Baithalu, R.K., and Mohanty, T.K. (2018). Transcriptional abundance of type-1 endocannabinoid receptor (CB1) and fatty acid amide hydrolase (FAAH) in bull spermatozoa: Relationship with field fertility. Theriogenology 114, 252–257.
Lee, M.S., Lanes, A., Ginsburg, E.S., and Fox, J.H. (2020). Delta-9 THC can be detected and quantified in the semen of men who are chronic users of inhaled cannabis. J. Assist. Reprod. Genet.
López-Cardona, A.P., Ibarra-Lecue, I., Laguna-Barraza, R., Pérez-Cerezales, S., Urigüen, L., Agirregoitia, N., Gutierrez-Adán, A., and Agirregoitia, E. (2018). Effect of chronic THC administration in the reproductive organs of male mice, spermatozoa and in vitro fertilization. Biochem. Pharmacol.
Martínez-León, E., Osycka-Salut, C., Signorelli, J., Kong, M., Morales, P., Pérez-Martínez, S., and Díaz, E.S. (2018). Fibronectin modulates the endocannabinoid system through the cAMP/PKA pathway during human sperm capacitation. Mol. Reprod. Dev.
Migliaccio, M., Ricci, G., Suglia, A., Manfrevola, F., Mackie, K., Fasano, S., Pierantoni, R., Chioccarelli, T., and Cobellis, G. (2018). Analysis of endocannabinoid System in Rat Testis During the First Spermatogenetic Wave. Front. Endocrinol. 9, 269.
Miller, M.R., Mannowetz, N., Iavarone, A.T., Safavi, R., Gracheva, E.O., Smith, J.F., Hill, R.Z., Bautista, D.M., Kirichok, Y., and Lishko, P.V. (2016). Unconventional endocannabinoid signaling governs sperm activation via sex hormone progesterone. Science.
Murphy, S.K., Itchon-Ramos, N., Visco, Z., Huang, Z., Grenier, C., Schrott, R., Acharya, K., Boudreau, M.-H., Price, T.M., Raburn, D.J., et al. (2018). cannabinoid exposure and altered DNA methylation in rat and human sperm. Epigenetics.
Nassan, F.L., Arvizu, M., Mínguez-Alarcón, L., Williams, P.L., Attaman, J., Petrozza, J., Hauser, R., Chavarro, J., and EARTH Study Team (2019). Marijuana smoking and markers of testicular function among men from a fertility centre. Hum. Reprod. Oxf. Engl.
Nielsen, J.E., Rolland, A.D., Rajpert-De Meyts, E., Janfelt, C., Jørgensen, A., Winge, S.B., Kristensen, D.M., Juul, A., Chalmel, F., Jégou, B., et al. (2019). Characterisation and localisation of the endocannabinoid system components in the adult human testis. Sci. Rep. 9, 12866.
Ückert, S., la Croce, G., Bettiga, A., Albrecht, K., Buono, R., Benigni, F., Kuczyk, M.A., and Hedlund, P. (2017). Expression and distribution of key proteins of the endocannabinoid system in the human seminal vesicles. Andrologia.
Whan, L.B., West, M.C.L., McClure, N., and Lewis, S.E.M. (2006). Effects of delta-9-tetrahydrocannabinol, the primary psychoactive cannabinoid in marijuana, on human sperm function in vitro. Fertil. Steril. 85, 653–660.
Zufferey, F., Donzé, N., Rahban, R., Senn, A., Stettler, E., Rudaz, S., Nef, S., and Rossier, M.F. (2020). Semen endocannabinoids are correlated to sperm quality in a cohort of 200 young Swiss men. Andrology.