Insights into the adverse effects of prepubertal chronic ethanol exposure on adult female reproduction

Prepubertal ethanol exposure in a female mouse model

Three-week-old C57BL/6J female mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. The mice were raised in cages of five and divided into two groups. One was the control group (CtrL), the other was the 3-week ethanol exposure group (EtOH) by replacing drinking water with 20% v/v ethanol.

Glucose tolerance test and insulin resistance test

The blood glucose of all mice was measured, and the mean-values of blood glucose of CtrL- and EtOH- groups were calculated, respectively. The mice with blood glucose levels near mean-value were selected for testing. For glucose tolerance test (GTT) and insulin resistance test (ITT), mice were fasted for 16 h and 6h, respectively, then 2 g/kg glucose or 1 IU/kg insulin was injected in the abdominal hypodermis. Tail blood was collected for blood glucose detection and blood glucose was measured 0 min, 15 min, 30 min, 45 min, 60 min, 90 min and 120 min after injection by a glucometer (Roche).

Estrous cycle detection

After a 3-week treatment, mice were evaluated for estrous cycles daily for 18 days at the same time by vaginal lavage. The first 6 days of vaginal lavage were the time of acclimation for the mice, the other 12 days of vaginal lavage were used for smears. The smears were observed under a microscope, and classified as proestrus (P), estrus (E), metestrus (M) and diestrus (D) through the proportion of nucleated epithelial cells, cornified squamous epithelial cells and leukocytes [17].

Oocyte maturation, fertilization and embryonic development

The processes followed published protocols [18]. The ovaries were removed with tweezers and weighed, then chopped up in M2 medium (Sigma, USA). The GV-stage oocytes were selected microscopically and matured in vitro in M2 medium. The number of germinal vesicle breakdown (GVBD) oocytes was recorded after 2-4 hours and the number of oocytes that extruded polar bodies (PBE) was recorded after 14 hours. Forty-eight hours after i.p. injection with pregnant mare serum gonadotrophin (Ningbo, China), hCG (Ningbo, China) was injected. Fourteen hours later, cumulus-oocyte complexes (COCs) were collected from the Fallopian tubes. For IVF, spermatozoa were obtained from healthy adult male mice and kept in HTF (Merck, Germany) for 40 min. Sperm and COCs were incubated in HTF. After 10-20 minutes of incubation, MII-stage oocytes with normal morphology were selected microscopically and then co-incubated with spermatozoa for 4-6h in HTF. After sperm-oocyte co-incubation, embryos were cultured in KSOM (Millipore, Germany) until development to blastocysts in vitro. The fertilization rates were determined by counting the number of pronuclei observed 8-9 hours post-fertilization. The number of blastocysts was confirmed at 4.5dpc and 5.5dpc respectively. In addition, female mice with vaginal plug after mating with male were used to observe blastocysts in vivo at 3.5 dpc by uterus flushing.

Fertility determination

After three weeks of treatment, the female mice were discontinued ethanol exposure, and mated with healthy adult males for 5 days. The dates of females secreting vaginal plugs were recorded. The pregnancy in the mice with vaginal plugs was recorded to calculate the fertility rate, and fetuses and placentas were collected intact using scissors and forceps for weighing at 19.0 dpc.

Chromosome spreading

The processes were performed as described previously [19]. MII-stage oocytes matured in vivo were collected 14 hours later from the Fallopian tubes, and digested with 1 mg/ml hyaluronidase (Sigma, USA). The zona pellucida of MII-stage oocytes matured in vivo were removed with acid Tyrode’s solution (Sigma, USA). The oocytes without zona pellucida were fixed in 1% paraformaldehyde with 0.15% Triton X-100 and 3 mM dithiothreitol on glass slides. The slides were dried at room temperature and blocked with 1% BSA for 1 h, then incubated in anticentromere antibodies, (ACA; 1:50) at 4° C overnight. After washing, the slides were incubated with secondary antibodies Cy5 (1:200) for 1 h at room temperature. The nuclei were stained with DAPI at room temperature for 15 min. DABICO was added and coverslips were used for lightly covering the samples.

Immunofluorescence

MII-stage oocytes were collected and fixed in 4% paraformaldehyde for 20 min. After incubation in 0.1% Triton X-100 for 20 min, the oocytes were transferred to 1 mg/ml BSA for 1 h. The processes above were performed at room temperature. Then the oocytes were incubated with α-tubulin antibody at 4° C overnight, and nuclei were stained with DAPI. Finally, samples were placed on the slides and DABICO was applied. All the specimens were observed under a Zeiss LSM 780 confocal laser-scanning microscope.

RNA-seq

RNA-seq was performed by Annoroad Gene Tech. (Beijing) Co., Ltd. The MII-stage oocytes were collected by superovulation from 3-week-old mice, 6-week-old control mice and ethanol-exposed mice in tubes with lysing reagent (30 oocytes per tube). All the collected oocytes had obviously extruded polar bodies. Ethanol-treatment did not stop during superovulation in the ethanol-exposed group. Amplification was conducted by the Smart-Seq2 method. After library preparation, PerkinElmer LabChip GX Touch and Step OnePlus Real-Time PCR System were introduced for library quality inspection, and PE150 sequencing was performed using the Illumina HiSeq platform. The experiments of each group were repeated three times. The RNA-seq data were available under NCBI SRA accession number PRJNA719111.

Statistical analysis

Data from repeated experiments were analyzed by independent sample T test by SPSS software. P<0.05 was considered a significant difference. The area under the curve (AUC) of GTT and ITT were calculated with GraphPad Prism version 6.01 for windows, GraphPad Software, La Jolla California USA. Correlation analysis was performed on the gene expression levels of each group by SPSS.

Establishment of prepubertal chronic ethanol exposure mouse model

To understand the effect of prepubertal chronic ethanol exposure on adult reproduction, we established a prepubertal ethanol exposure female mouse model by changing the 3-week-old mice’s drinking water to 20% v/v ethanol for 3 weeks. After a 3-week ethanol treatment, mice went through prepuberty, and the weight of ethanol-treated mice (n=11) was significantly lower than that of the control mice (n=16) (Figure 1A).

Figure 1. Establishment of prepubertal chronic ethanol-exposed mouse model. (A) Body weight. (B) Blood glucose levels in glucose tolerance test (GTT). (C) Blood glucose levels in insulin resistance test (ITT). (D) Estrous cycle. P, proestrus, E, estrus; M/D, metestrus and diestrus; *, p<0.05. ***, p<0.001.

The GTT and ITT were also performed to make comparison between the control and ethanol-exposed groups. GTT showed that the glucose level at 30-min post-injection was significantly higher in ethanol-treated mice (16.13 ± 0.6438 mmol/L vs 13.50 ± 0.4359 mmol/L, p<0.05; n=6 vs 6) (Figure 1B), and ITT showed that the glucose level at 90-min post-injection of insulin was also significantly higher in ethanol-treated mice (12.03 ± 0.8950 mmol/L vs 7.133 ± 0.7881 mmol/L, p<0.05; n=6 vs 6) (Figure 1C). Although blood glucose of GTT and ITT changed at individual time points, the trend of change was not significant. The area under the curve (AUC) of GTT (1235 ± 52.12 mmol/L/min vs 1299 ± 22.26 mmol/L/min, p>0.05) and ITT (835.5 ± 42.11 mmol/L/min vs 962.3 ± 58.14 mmol/L/min, p>0.05) of both groups were almost the same. The peak value and the corresponding time point of both groups showed no significant difference on GTT and ITT. The risks of prepubertal ethanol exposure cannot be measured by blood sugar. In other words, the absence of abnormal blood sugar levels does not mean that alcohol is not harmful to health.

To further understand the state of prepubertal ethanol exposed mice, the estrus cycle detection was carried out after 3-week of ethanol exposure by vaginal smears. Vaginal smears were examined for 18 consecutive days. The first 6 days of vaginal lavage were the time of acclimation for the mice, the other 12 days of smears were used to record the estrus cycle of model mice (n=17) and control mice (n=16). The results showed that the prepubertal ethanol-exposed female mice had a disordered estrus cycle, and most of the time the ethanol-exposed mice were in metestrus and diestrus (Figure 1D). This suggested that prepubertal chronic ethanol exposure might have adverse reproductive effects in adults.

Effects of prepubertal chronic ethanol exposure on adult female fertility

After a 3-week ethanol exposure, the mice reached puberty. The ethanol exposure was stopped immediately, and the mice were mated with normal adult males. Females were observed daily for the presence of vaginal plugs, and mice with vaginal plugs were recorded as successful mating. The production of offspring was recorded as being fertile. The results showed that the fertility rate of prepubertal ethanol-exposed female mice decreased significantly compared with control mice (Figure 2A). After discontinuation of ethanol exposure, the distribution of successful mating dates was shown in Figure 2B. A day or two after abstaining from ethanol, fewer mice mated successfully and produced few offspring. The fertility rate of the successfully mating mice on day 3 after abstaining from ethanol was significantly lower than that of the control mice, and that of day 3 and 4 showed no significant difference between the two groups (Figure 2C). Weight of placentas collected at 19.0 dpc showed little difference in prepubertal ethanol-exposed mice that mated successfully on day 4 and 5 compared to the control mice (Figure 2D). Fetal weight of prepubertal ethanol-exposed mice mating successfully on day 4 was significantly lower than that of the control mice, and the difference was eliminated at day 5 (Figure 2E). From this, we inferred that ethanol has an adverse effect on mouse reproduction, and timely withdrawal of ethanol exposure restore fertility to some extent. It was unknown whether the adverse effect on mouse reproduction occurred only during fertilization and embryonic development or began at the oocyte stage.

Figure 2. Effects of prepubertal chronic ethanol exposure on fertility. (A) the fertility rate of prepubertal ethanol-exposed female mice. (B) The distribution of successful mating dates after discontinuation of ethanol exposure. (C) The fertility rate of prepubertal ethanol-exposed mice from different successful mating dates. (D) Placenta weight of prepubertal ethanol-exposed mice from different successful mating dates. (E) Fetus weight of prepubertal ethanol-exposed mice from different successful mating dates. *, p<0.05; **, p<0.01.

Prepubertal chronic ethanol exposure leads to a decrease in oocytes of adult mice

To understand the effects of prepubertal chronic ethanol exposure on female reproduction, the examination of oocyte number and quality of prepubertal ethanol-exposed mice is necessary. The results showed that the ovarian weight of prepubertal ethanol-exposed mice was significantly reduced (Figure 3A). In vitro culture of GV-stage oocytes taken from ovaries showed no significant difference in GVBD rates, but the PBE rate was significantly reduced (Figure 3B, 3C). The number of normal MII-stage oocytes collected by superovulation from each prepubertal ethanol-exposed mice (n=15) was significantly lower than that of the control group (n=10) (Figure 3D). Therefore, we inferred that ethanol feeding leads to abnormal growth and development of ovary and reduces the number of oocytes.

Figure 3. Effects of prepubertal chronic ethanol exposure on ovary and oocytes. (A) Ovary weight. (B) Germinal vesicle breakdown (GVBD) rate. (C) Polar body extrusion (PBE) rate. (D) Number of MII-stage oocytes in vivo. (E) Chromosome spreading in MII-stage oocytes matured in vivo. (F) Spindle stained in MII-stage oocytes matured in vivo.

Prepubertal ethanol exposure causes reduced fertilization ability of morphologically normal adult oocytes, but once oocytes fertilized have the ability to develop into blastocysts

It was reported that oocytes exposed to ethanol solution might show chromosome separation abnormalities by affecting the spindle [20, 21]. In our study, ethanol exposure caused abnormal morphology in some oocytes, while the other oocytes still showed normal morphology whose status remained uncertain. Immunostaining showed that spindles were basically normal and the separation of chromosomes was normal in morphologically normal oocytes with extruded first polar body (Figure 3E, 3F). We next clarified whether ovulated oocytes with normal morphology from ethanol exposed mice have normal developmental potential. IVF was performed for MII-stage oocytes with normal morphology obtained by superovulation. Prepubertal ethanol-exposed groups showed a significant decrease in the fertilization rate (Figure 4A), but no significant difference in blastocyst rate was observed when counting from zygotes to blastocysts (Figure 4B). After superovulation, both in vitro and in vivo matured oocytes had the ability to produce normal blastocysts (Figure 4C). This suggested that oocytes with normal morphology from ethanol exposed mice, once fertilized, could develop into blastocysts, although prepubertal ethanol exposure had a negative effect on the maturation and fertilization of oocytes.

Figure 4. Effects of prepubertal chronic ethanol exposure on fertilization ability. (A) Fertilization rate after in vitro fertilization. (B) Blastocyst rate from zygotes. (C) The morphology of blastocysts produced in vitro and in vivo. ACA, anticentromere antibodies; *, p<0.05; ***, p<0.001. (D) Bivariate analysis between any two of CtrL-3-weeks, CtrL-6-weeks, and EtOH-6-weeks.

RNA-seq for ethanol exposed oocytes with normal morphology

To determine the quality of the oocytes with normal morphology after ethanol exposure during the period from 3 to 6 weeks after birth, RNA-seq was performed on MII-stage oocytes from control mice (CtrL-6-week) and the oocytes with normal MII-stage morphology from ethanol exposed mice (EtOH-6-week). Comparing CtrL-6-week-group and EtOH-6-week-group, GO analysis showed that there were 234 GO terms which were significantly enriched (P<0.05, Figure 5A). KEGG analysis showed that there were 8 KEGG pathways which were significantly enriched (P<0.05, Figure 5B). Among them, topping the list of KEGG pathways is “Cell cycle” pathway. The results showed that alcohol had an adverse effect on oocytes related to cell cycle, meiosis and cell senescence and other factors.

Figure 5. GO analysis and KEGG analysis between CtrL-6-week-group and EtOH-6-week-group. (A) GO analysis. (B) KEGG analysis.

The GO and KEGG analysis showed that the state of oocytes was changed by ethanol treatment, but the intensity of the change was difficult to predict. To understand the damage caused by 3 weeks of ethanol treatment, RNA-seq was performed on oocytes from normal female mice (CtrL-3-week) at 3 weeks of age without ethanol treatment and sexual maturation. Bivariate analysis was used to analyze the correlation between any two of CtrL-3-week, CtrL-6-week, and EtOH-6-week (Figure 4D). Some studies have shown that the developmental potential of oocytes before puberty was lower than that after sexual maturation [15, 16], this explained the relatively low correlation coefficient between CtrL-3-weeks and CtrL-6-weekss (Pearson coefficient = 0.840). The correlation coefficient between CtrL-3-weeks and CtrL-6-weeks was lower than that between EtOH-6-weeks and CtrL-6-weeks (Pearson coefficient = 0.919), suggesting that the damage to morphologically normal oocytes after ethanol treatment was weaker than the lack of developmental potential caused by insufficient sexual maturation. In addition, The correlation coefficient between CtrL-3-weeks and CtrL-6-weeks was lower than that between EtOH-6-weeks and CtrL-3-weeks (Pearson coefficient = 0.907). It could be speculated that there might be some correlation between ethanol injury and the loss of developmental potential without experiencing sexual maturation. These results might help us to understand the extent of oocyte damage caused by ethanol.