Effects of Delta-9 Tetrahydrocannabinol (THC) on Oocyte Competence and Early Embryonic Development

Abstract

Recent changes in legal status and public perception of cannabis have contributed to an increase use amongst women of reproductive age. Concurrently, there is inadequate evidence-based knowledge to guide clinical practice regarding cannabis and its effects on fertility and early embryonic development. This study aimed to evaluate the effects of the primary psychoactive component of cannabis, delta-9 tetrahydrocannabinol (THC), during oocyte maturation, and its impact on the developing embryo. Bovine oocytes were matured in vitro for 24 h under clinically relevant doses of THC mimicking plasma levels achieved after therapeutic (0.032 μM) and recreational (0.32 and 3.2 μM) cannabis use. THC-treated oocytes were assessed for development and quality parameters at both the oocyte and embryo level. Characteristics of oocytes treated with cannabinoid receptor antagonists were also assessed. Oocytes treated with 0.32 and 3.2 μM THC, were significantly less likely to reach metaphase II (p < 0.01) and consequently had lower cleavage rates at day 2 post-fertilization (p < 0.0001). Treatment with cannabinoid receptor antagonists restored this effect (p < 0.05). Oocytes that did reach MII showed no differences in spindle morphology. Oocytes treated with 0.032 μM THC had significantly lower connexin mRNA (p < 0.05) (correlated with decreased quality), but this was not confirmed at the protein level. At the blastocyst stage there were no significant differences in developmental rates or the proportion of trophectoderm to inner cell mass cells between the control and treatment groups. These blastocysts, however, displayed an increased level of apoptosis in the 0.32 and 3.2 μM groups (p < 0.0001). Our findings suggest a possible disruptive effect of cannabis on oocyte maturation and early embryonic development.

Keywords: THC; cannabis; embryo; fertility; oocyte.

Figure 1

Developmental parameters after oocyte THC exposure. Oocytes were matured in varying doses of THC mimicking therapeutic cannabis use (low, 0.032 μM) or recreational cannabis use (mid, 0.32 μM; high, 3.2 μM), in addition to a control and vehicle (1:1:18 ethanol: tween: saline) groups. (A) Oocyte maturation rate. Oocytes were denuded from their cumulus cells after 24 h maturation and the presence of the first polar body was assessed as a determinant of a metaphase II state. The rate was calculated over the total number of oocytes. Three biological replicates of at least 50 oocytes/group was carried out, totalling 164 oocytes/group. (B) Cleavage rate. After 22 h maturation, oocytes were fertilized and cultured. The cleavage rate was assessed at 48 h post-fertilization and calculated over the number of total oocytes cultured. Eleven biological replicates consisting of at least 60 oocytes/group were analyzed for a total of 836 oocytes/group. (C) Blastocyst rate over the total number of oocytes. Embryos were cultured and rate of blastocyst development was measured at day 8 post-fertilization. Seven biological replicates of at least 60 oocytes/group were analyzed with a total of 507 oocytes/group used (D) Blastocyst rate calculated over cleavage rate. Bars represent mean ± SEM. Significance is calculated compared to vehicle group. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 2

Spindle morphology assessment in metaphase II oocytes. (A) Oocytes with grade A spindles. (B) Oocytes with grade B spindles. (C) Oocytes with grade C spindles. (D) Proportion of each oocyte grade per treatment group. Oocytes were matured in media containing THC doses representing therapeutic cannabis use [low (THC) (0.032 μM)] and recreational cannabis use [mid (THC) (0.32 μM), high (THC) (3.2 μM)] in addition to a vehicle and control group for 24 h. Following denuding, oocytes displaying polar body extrusion were deemed at metaphase II and underwent immunocytochemistry staining for spindles. Spindle and chromosome morphology were graded and categorized, with grade A oocytes having chromosomes aligned on the metaphase plate and organized spindles. Grade C oocytes had misaligned chromosomes and disorganized spindles. Seventeen replicates of at least 5 oocytes/group were examined for a total of 70 and 83 oocytes/group. Bars represent the proportion of oocytes in each group.

Figure 3

Differential staining of blastocysts produced from THC treated oocytes. (A) Control blastocyst. (B) Vehicle blastocyst. (C) Low (THC) (0.032 μM) blastocyst. (D) Mid (THC) (0.32 μM) blastocyst. (E) High (THC) (3.2 μM) blastocyst. (F) Proportion of trophectoderm (TE) vs. inner cell mass (ICM) cell numbers for each group. Oocytes underwent maturation in varying doses of THC representing therapeutic cannabis use [low (THC)] and recreational cannabis use [mid (THC), high (THC)] as well as vehicle or control, followed by fertilization and culture. Blastocysts were collected at 8 days post-fertilization and differentially stained. The trophectoderm (TE) was stained with propidium iodide (pink) and inner cell mass (ICM) with Hoechst stain (blue). Blastocysts were imaged with a Leica CTR5500B fluorescent microscope at the 20 × objective and cells manually counted. Each group had between 4 and 11 biological replicates (blastocysts). Bars represent the mean ± SEM. Scale bar =100 μm.

Figure 4

Total nuclei count in blastocysts produced from THC treated oocytes. (A) Control blastocyst. (B) Vehicle blastocyst. (C) Low (THC) (0.032 μM) blastocyst. (D) Mid (THC) (0.32 μM) blastocyst. (E) High (THC) (3.2 μM) blastocyst. (F) Total cell number. Oocytes were matured in doses of THC correlating to therapeutic cannabis use [low (THC)] and recreational cannabis use [mid (THC), high (THC)] as well as vehicle and control groups, followed by fertilization and culture to the blastocyst stage. Blastocysts were collected at 8 days post-fertilization and stained using either the differential staining or TUNEL protocol. Blastocysts were imaged with a Leica CTR5500B fluorescent microscope at the 20 × objective. Cells were counted manually and verified using ImageJ software. Each group had 25–34 biological replicates (blastocysts) analyzed. Bars represent the mean ± SEM. Scale bar =100 μm.

Figure 5

Connexin mRNA expression in cumulus-oocyte complexes (COCs) and blastocysts. (A) Normalized expression of connexin 37 (CX37) in 250 ng of total mRNA from 15 COCs to reference genes YWHAZ and GAPDH. (B) Normalized connexin 43 (CX43) expression in 250 ng of mRNA from 15 COCs. (C) Normalized expression of CX37 in 5 blastocysts. (D) Normalized expression of CX43 in 5 blastocysts. COCs were collected after 22 h of maturation in the treatment groups or fertilized, cultured and collected as blastocysts at day 8 post-fertilization. Total RNA was extracted and connexin mRNA was quantified using droplet digital PCR. Nine biological replicates were carried out in COCs and 5 biological replicates in blastocysts. Bars represent mean ± SEM. Significance is calculated in relation to the vehicle group. *p < 0.05, **p < 0.01.

Figure 6

Expression of connexin protein in cumulus-oocyte complexes (COCs). (A) Western blot image of connexin 37 (CX37) and the corresponding GAPDH as the loading control. (B) Densitometric analysis of CX37 protein expression relative to GAPDH. (C) Western blot image of connexin 43 (CX43) and corresponding GAPDH. (D) Densitometric analysis of CX43 and relative GAPDH protein. Proteins were extracted from groups of 40 COCs after 22 h of maturation in one of the five treatment groups. Six biological replicates for CX37 and 3 biological replicates for CX43 were analyzed. Bars represent mean ± SEM.

Figure 7

TUNEL staining of blastocysts. (A) Negative control blastocyst. (B) Positive control blastocyst (DNase I-treated). (C) Control blastocyst. (D) Vehicle blastocyst. (E) Low (THC) (0.032 μM) blastocyst. (F) Mid (THC) (0.32 μM) blastocyst. (G) High (THC) (3.2 μM) blastocyst. (H) Average DNA fragmentation over total number of nuclei. Cumulus-oocyte complexes (COCs) were matured in either THC, vehicle or control media followed by fertilization and culture for 8 days when blastocysts were collected. Blastocysts underwent TUNEL staining which stained nuclei undergoing DNA fragmentation with FITC label (green) and all nuclei with Hoechst stain (blue). Red arrows in (C–G) point out apoptotic cells. Blastocysts were imaged with a Leica CTR5500B fluorescent microscope at the 20 × objective. Between 19 and 26 total blastocysts per group were analyzed. Bars represent the mean ± SEM. Significance is calculated compared to vehicle group. Scale bar = 100 μm. **p < 0.01, ***p < 0.0001.

Figure 8

Caspase 9 mRNA expression in blastocysts. Blastocysts were produced from cumulus-oocyte complexes (COCs) exposed to varying doses of THC representing therapeutic cannabis use (low, 0.032 μM) and recreational cannabis use (mid, 0.32 μM; high, 3.2 μM) in addition to a vehicle (1:1:18 ethanol: tween: saline) and control group for 22 h. COCs were fertilized and cultured to the blastocyst state. At day 8 post-fertilization, blastocysts were collected in groups of 5 and underwent RNA extraction followed by droplet digital PCR quantification of caspase 9 (cas-9). Results were normalized to the reference gene, YWHAZ. Four biological replicates were analyzed. Bars represent mean ± SEM. Significance is calculated in relation to the vehicle group. *p < 0.05.

Figure 9

Developmental parameters of oocytes treated with THC and a selective cannabinoid antagonist. (A) Cleavage rate over total number of oocytes. Nine biological replicates of at least 60 oocytes/group were analyzed totalling 586 oocytes/group. (B) Blastocyst rate over total number of oocytes. Seven biological replicates of at least 60 oocytes/group was used for a total of 479 oocytes/group. (C) Blastocyst rate calculated over cleavage rate. Following the standard in vitro embryo production procedure, oocytes were matured in one of five groups: control, vehicle (1:1:18 ethanol:tween:saline), high (THC) (3.2 μM), CB1 antagonist (3.2 μM THC + 3.2 μM SR141716) and CB2 antagonist (3.2 μM THC + 3.2 μM SR144528). Mature cumulus oocyte complexes (COCs) were fertilized and cultured with the cleavage rate assessed at 48 h post-fertilization and blastocyst rate at day 8 post-fertilization. Bars represent the mean ± SEM. Significance is calculated compared to vehicle group. *p < 0.05, **p < 0.01, ****p < 0.0001.