Effect of vitamin K and co­enzyme Q on cleavage rates and developmental potential of mice embryos in an in vitro fertilization setting

Kezia Emeny­Smith and Taylor Reynolds

California Polytechnic State University, San Luis Obispo, California March 10, 2015

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Abstract

During preimplantation embryonic development, the mitochondrion undergoes significant structural and functional changes [1]. Mitochondria play important roles within the body, including ATP generation and many other cellular processes; therefore, mitochondrial function contributes to embryo developmental confidence [1]. When used to supplement standard in vitro culture media, vitamin K and co­enzyme Q have been shown to repair mitochondrial damage, thus improving embryo development potential [2,3]. The present study compares cleavage rates and developmental potential of mice embryos with or without the addition of vitamin K and co­enzyme Q to the culture media. The results show no significant difference in developmental potential when vitamin K and co­enzyme Q are added to the fertilization media.

Introduction

In vitro fertilization (IVF) is an assisted reproductive technology in which fertilization is performed outside of the body, typically in a dish or tube, and requires sufficient oocyte maturation, sperm separation, and sperm capacitation. In animals, IVF is mainly used for research purposes but can also be used in cases of abnormalities, semen sexing, to address limited sperm supply, to fertilize multiple eggs at once, and to overcome poor sperm motility. The present study was conducted to determine if the addition of vitamin K and co­enzyme Q to in vitro culture media improves cleavage rates and developmental potential of mice embryos.

Mitochondria are organelles that play a role in normal cell function, such as apoptosis regulation, lipid metabolism, steroid synthesis, calcium homeostasis, and energy production [1]. Currently, their role in ATP production is the most investigated topic; however, it has become apparent that mitochondria play a significant role in the developmental potential of oocytes and preimplantation stage embryos [4]. Creating a culture media that replicates the in vivo environment has been attempted but in vitro conditions disrupt adequate mitochondrial function, causing embryos produced in vitro to be of lower quality [1,4]. Previous studies using bovine embryos have shown that the manipulation of oocyte conditions within culture media improves blastocyst formation rates and expression of genes related to mitochondrial function [5].

Mitochondria are maternally inherited and create a balance between ATP production by the electron transport chain and oxidative stress. Preimplantation embryos rely on maternally inherited mitochondria for energy to develop [6]. In vitro conditions often result in compromised mitochondrial function in embryos; however, the presence of co­enzyme Q and vitamin K in culture media has been shown to rescue mitochondrial function. Vitamin K acts as a membrane­bound mitochondrial electron carrier during ATP production by the electron transport chain, and co­enzyme Q is an electron carrier crucial for electron transfer within the mitochondrial membrane and plays a role in protecting biological membranes from oxidative damage [3]. Vitamin K and co­enzyme Q are electron carriers that aid in the movement of protons throughout the electrochemical gradient of the electron transport chain. When supplemented with both of these electron carriers, the electron transport chain ultimately produces more ATP, thus increasing available energy for embryos to utilize in their preimplantation stage development [3]. Previous studies using bovine models show that addition of vitamin K to culture media is most effective when done 72 hours after fertilization, resulting in embryos of higher morphological quality, and significantly increasing the percentages of blastocysts and expanded blastocysts [2]. Additionally, vitamin K supplementation significantly improved mitochondrial activity and had a notable influence on embryonic gene expression [2].

Based on the previous study performed by Baldoceda, et al. 2014 [2], and the similar function of coenzyme Q to vitamin K, we hypothesized that the addition of vitamin K and co­enzyme Q to culture media at 72 hours post­fertilization would increase cleavage rates and development of mice embryos to the blastocyst stage. In the present study, we intended to

investigate if the addition of vitamin K and coenzyme Q at 72 hours after the in vitro fertilization of mouse embryos would improve embryonic mitochondrial function and result in more developmentally competent embryos when compared to embryos cultured in non­supplemented media. 2) Material and Methods: 2.1. Superovulation and Animal Sacrificing

Mature female mice (3 per trial, 4 trials total) were each injected with 0.1ml pregnant mare’s serum gonadotropin (PMSG) 67.5 hours pre­fertilization (Table 3) to induce follicular growth, mimicking the effect of the body’s natural hormone, FSH. Exactly 47 hours after receiving PMSG (Table 3), female mice received 0.1ml human chorionic gonadotropin (hCG) to induce ovulation, mimicking the effect of the body’s natural hormone, LH. All injections were administered intraperitoneally using 25 gauge needles and 1 ml syringes. 20.5 hours post­hCG injection, the 3 female mice and 1 male mouse were humanely euthanized via carbon dioxide administration in an induction box, followed by cervical dislocation. Animal handling and euthanasia were performed in accordance with California Polytechnic State University Institutional Animal Care and Use Committee guidelines. 2.2. Collection and Preparation of Gametes

A. Female The ovaries and the oviduct of each female mouse were excised and placed in a 60mm

dish containing G­MOPS™ Plus, and the oocytes were collected from the oviduct using a 25 gauge needle. The oocytes were washed and sorted in six 30 µl drops of pre­warmed G­MOPS™ Plus holding media under mineral oil. A total of four trials were performed.

B. Male The testis and a portion of the vas deferens were excised and placed in a 60mm dish

containing G­MOPS™ Plus, where the epididymis was removed. Spermatozoa from the caudal epididymis were placed in a sterile tube of 500 µl pre­warmed G­IVF™ Plus media. The sperm were incubated and underwent a swim­up for a minimum of thirty minutes. Once complete, a 400 µl supernatant was removed and placed into a new, sterile tube for fertilization. A total of four trials were performed. Due to consistently low sperm concentrations, hemocytometer procedures were excluded from the all four trials.

2.3. In Vitro Fertilization

Following washing of collected oocytes in G­MOPS™ Plus, oocytes were placed in a 30 µl drop of pre­incubated G­IVF™ Plus media under mineral oil. 10 µl of spermatozoa were then added to the G­IVF™ Plus drop for fertilization. Oocytes and sperm were co­incubated at 37.5 degrees Celsius with 5% CO2. A total of four trials were performed.

2.4. Embryo Culture One day post­in vitro fertilization, presumptive zygotes were transferred to G­1™ Plus

media and incubated at 37.5° Celsius under 5% CO2. After 2 days of culture, embryos were sorted into a control and treatment group and evaluated for cleavage. Half of the embryos were placed in a new culture dish containing a 30µl drop of G­2™ Plus media and half of the embryos were placed in a new culture dish containing a 30µl drop of G­2™ Plus media with the addition of Vitamin K and Co­enzyme Q. On day 4 post­fertilization, embryos were evaluated for stage of development using the International Embryo Transfer Society guidelines. 2.5 Assessment of Fertilization and Quality of Embryos

Embryos were assessed for fertilization and normality one day post­in vitro fertilization. Normal fertilization was defined as the presence of 2 pronuclei in the ooplasm. Any embryos that deviated from these requirements (abnormal or unfertilized) were discarded from the trial and excluded from further analysis. Stage­development and quality grading of embryo was done using the International Embryo Transfer Society (IETS) guidelines (Table 1 and Table 2). Table 1. IETS Guidelines for Assessing Embryo Stage Development

Stage Stage of Development

1 Unfertilized

2 2­12 cells

3 Early morula

4 Morula

5 Early blastocyst

6 Blastocyst (blastocoel capsule formed, ICM formed)

7 Expanded blastocyst (ZP thins, ICM, and embryo is symmetrical)

8 Hatched blastocyst

9 Expanded blastocyst

Table 2. IETS Guidelines for Grading Embryo Quality

Grade Embryo Quality

1 Excellent/Good (<10% not good)

2 Fair (70% ok, 30% not good)

3 Poor (>30% not good)

4 Dead/Degenerating

2.6. Experimental Design The objective of the current experiment was to determine the effect of vitamin K and

co­enzyme Q on cleavage rate and developmental potential of mice oocytes. Drops containing day 3 embryos were incubated with or without vitamin K and co­enzyme Q added to the fertilization media, G­2™ Plus. The experiment was replicated a total of four times. Table 3. Experiment Procedural Timeline

Sunday 9:30PM

Tuesday 8:30PM

Wednesday 5­7PM

Thursday 11AM­1P

M

Saturday 10­11AM

Sunday 11AM

PMSG 3 hCG 3

∙ Sacrifice 3 & 1 ∙ IVF

Check for pronuclei

∙ Check 2­cell cleavage ∙ Add CoQ/Vit. K to trial group

∙ 8­cell cleavage ∙ Assess

3) Results

Oocytes (n=155) were retrieved from twelve superovulated female mice throughout four trials. During the four trials, a total of sixty­five oocytes (41.9%) were fertilized one day after the in vitro fertilization procedure (Table 4). This corresponds to approximately 13 oocytes retrieved per female mouse. Fertilization rates were calculated by dividing the number of fertilized oocytes day 1 post in vitro fertilization by the total amount of collected oocytes for each trial. Embryos were assessed 24 hours, 72 hours, and 96 hours post­in vitro fertilization for development and cleavage rate. At 96 hours post­in vitro fertilization, embryos were analyzed and assigned a stage and grade for each treatment group (Figure 3 and 4). In trial 1, 4 embryos were assigned to the treatment group and 1/ 4(25%) remained unfertilized, while 3/ 4 (75%) were stage 2, grade 4 embryos. In trial 1, 3 embryos were assigned to the control group and 1/ 3 embryos progressed to the blastocyst stage, while 2/3 (66.7%) were stage 2, grade 4 embryos. In trial 2, 14 embryos were assigned to the treatment group and 14/14 (100%) were stage 2, grade 4 embryos. In trial 2, 13 embryos were assigned to the control group and 13/13 (100%) embryos were stage 2, grade 4 embryos. In trial 3, 6 embryos were assigned to the treatment group and 6/6 (100%) were stage 2, grade 4 embryos. In trial 3, 7 embryos were assigned to the control group and 7/7 (100%) embryos were stage 2, grade 4 embryos. In trial 4, 9 embryos were assigned to the treatment group and 9 / 9 (100%) were stage 2, grade 4 embryos. In trial 4, 9

embryos were assigned to the control group and 1/ 9 (11%) remained unfertilized, while 8/ 9 (89%) were stage 2, grade 4 embryos. Figure 1. Stages of mouse preimplantation development (Saiz et al., 2012) [7]

Table 4. Fertilization Rates for Trial 1 ­ Trial 4

Trial # Presumptive Zygotes from Collected Oocytes, Day 1 Post­Fertilization

Fertilized (%)

1 7/16 43.8%

2 27/62 43.5%

3 13/26 50.0%

4 18/51 35.3% Average= 43.2%

Figure 3. Stage and grade of embryos 96h post­fertilization (control). Figure 4. Stage and grade of embryos 96h post­fertilization (treatment).

A. Stage 2, Grade 4 embryo 5 days post­in vitro fertilization B. Stage 1 embryo 5 days post­in vitro fertilization

4) Discussion

Vitamin K and co­enzyme Q are often added to culture medium to improve embryo developmental competence [2,3]. The objective of this study was to investigate if the addition of vitamin K and coenzyme Q at 72 hours post­in vitro fertilization of mouse embryos would improve embryonic mitochondrial function and result in more developmentally competent embryos when compared to embryos cultured in non­supplemented media. It was hypothesized that embryos cultured with vitamin K and co­enzyme Q may have increased mitochondrial function leading to increased cleavage rate and overall higher developmental potential [1­4]. However, in the present study, the addition of vitamin K and co­enzyme Q did not improve cleavage rates and embryo development. Reasons for the discrepancies between this study and previous successful studies are difficult to resolutely pinpoint. However, there are a wide array of potential causes for the present study’s deviation from the expected outcome.

Throughout the experimental set­up, there were several factors that could have contributed to the unexpected outcome of the study’s results. The fertilization media used throughout this study was the G­series from Vitrolife. When revising the guidelines provided by the manufacturer of the media, we found that it was suggested to pre­incubate media between 6 to 18 hours prior to use [8]. Instead, this study only incubated media for approximately 30­45 minutes before a transfer; however, during trial 1 the G­2 Plus plates were prepared and incubated 12 hours prior to transfer. This trial led to the only blastocyst formation throughout all four trials. Although this is not a largely significant result, it offers some evidence that pre­incubating media for an extended period of time can improve developmental potential of in vitro produced mice embryos. Although a direct correlation cannot be made based upon one incidence alone, it seems plausible that with longer equilibration times, media more closely mimics the uterine environment in terms of pH and temperature, leading to further embryo developmental progression. A previous study that was successful in obtaining a significant amount of blastocysts using vitamin K supplemented culture media, explained that oocytes were

fertilized with semen after 15­18 hours of incubation at 38.5° C containing 5% CO2 [2]. This evidence implies that equilibrating oocytes in culture media for an extended period of time enhances blastocyst rates. In addition, Vitrolife also recommends washing the embryos through a plate of G­MOPS™ Plus before transferring between medias [8]. Due to economic restraints this step was omitted from the present study; however, given the resources we believe this could enhance the outcome in future trials.

One factor of experimental set­up that we were extremely meticulous about was maintaining a warm environment for gametes while manipulating and transferring in vitro. Throughout all trials, mature oocytes were used for fertilization, meaning that the meiotic spindle was formed. Meiotic spindles are formed from microtubules and are extremely sensitive to temperature fluctuations [5]. If temperatures dropped below or went above the desirable 38.5°C, it is likely that the spindle could have disassembled, thereby negatively affecting fertilization rates and embryo developmental potential. In efforts to minimize this potential hazard, a heated glass microscope stage was utilized and a heating plate at 38.5°C was kept close to the workspace at all times in efforts to maintain the temperature of an in vivo environment and stabilize the meiotic spindle formation.

According to the Jackson Laboratory, when fresh mouse sperm is used for in vitro fertilization, the average fertilization rate is 65% [9]. Throughout all four trials, fertilization rates were much lower than this average, 43.8%, 43.5%, 50.0%, and 35.3%, respectively (table 4). Many factors could have contributed to the consistently low fertilization rates present throughout the present study. As mentioned previously, if the temperatures were not ideal throughout oocyte handling, the meiotic spindle could have been disassembled [5]. Throughout all four trials the cd1 strain of mice were used. It is reasonable to hypothesize that this strain could be low performing mice in terms of reproductive potential. All females ovulated mature oocytes during each trial, while sperm concentrations were consistently low. Given the resources, investigating this strain would be something to further investigate for future trials, especially male fertility potential. In total, 155 oocytes were collected from 12 mature, female mice averaging about 13 collected oocytes per mouse. In addition to low fertilization rates, there was a consistent issue with unnaturally low epididymal sperm counts from the male mice used in the trials. The mice used across the four trials were smaller than average, leading us to speculate that the male mice potentially had lower than normal testosterone levels, which could have had a detrimental effect on sperm production. In addition, when sperm were viewed under a high magnification scope, many abnormalities were present. In particular, many cytoplasmic droplets were observed, indicating immature spermatozoa [10]. Due to the consistently low sperm counts, our experiment was confounded on two fronts. In the first two trials, 1ml of G­IVF ™ Plus was used for the sperm swim­up and resulted in very low sperm counts when semen was analyzed using the hemocytometer. In an effort to increase sperm concentration, the following two trials utilized .5mL G­IVF ™ Plus for the swim­up. Although the reduction of the G­IVF ™ Plus volume hypothetically should have had a favorable effect on sperm concentrations, the reduction of

volume made it more difficult to remove the desirable supernatant produced by the swim­up. The hurdle posed by having to remove only the desired 400µl of supernatant from a 500µl total volume subsequently increased the difficulty in achieving the appropriate concentration of sperm to use for in vitro fertilization. Theoretically, the swim­up would have led to capacitated, viable sperm remaining at the top portion of the supernatant; however, the difficulty of removing the desired 400 ul of supernatant could have resulted in non capacitated sperm being utilized for fertilization, furthering less than ideal fertilization rates (table 4). Due to the excessively low sperm counts, we were unable to use the hemacytometer effectively to adjust sperm counts to the desired concentrations. We ultimately decided to pipette 400µl of supernatant into a new tube, and from this new tube fertilized each oocyte­containing drop with 10µl of non­concentration adjusted motile sperm. This unexpected hindrance to our experimental design must be considered as a possible influence on the less than ideal results of the present study. For future trials, it could be advisable to sacrifice an additional male for each trial in order to maximize the amount of sperm available to use for the fertilization of oocytes.

Throughout all four trials, embryo development did not conform to the desired outcome. Besides the one blastocyst formation during trial 1, all fertilized oocytes did not progress past the 2­16 cell stage (stage 2) when analyzed post­in vitro fertilization. Figure 1 illustrates the stages of mouse preimplantation development [5]. It was expected that on day 1 post­in vitro fertilization, embryos would be at a two cell stage, developing into blastocysts by day 4 post­in vitro fertilization [5]. However, the majority of embryos were stage 2, grade 4 embryos when assessed day 4 post­in vitro fertilization as indicated by figure 5A. Reasons for the discrepancies have been previously discussed.

In conclusion, the present study found that when vitamin K and co­enzyme Q are added to in vitro fertilization media, cleavage rates and embryo developmental potential are not improved.

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