Morphometric, Subcellular, In-Vitro Fertilization and Embryonic Developmental Assessment of Mouse Oocytes Produced by Anti-Inhibin Serum or PMSG Superovulation

Abstract

This study compared morphometric, subcellular characteristics, in-vitro fertilization (IVF) and embryonic developmental potential of Metaphase II (MII) mouse oocytes obtained from females superovulated with either Anti-Inhibin Serum-hCG (AIS-hCG) or PMSG-hCG. The oocyte’s quantity, quality, zona pellucida (ZP) thickness, perivitelline space (PVS), diameter, microtubules, F-actin, cortical granules (CGs), and mitochondria distribution were determined. The superovulation by using AIS-hCG resulted in a higher numbers of oocyte/donor compared to PMSG-hCG (P=0.002). There was no difference in morphologically normal and abnormal oocytes between the AIS-hCG or PMSG-hCG (P=0.425 and P=0.194, respectively). The morphometric measurements showed no difference in oocyte diameter between AIS-hCG and PMSG-hCG (P=0.289). However, the thickness of the ZP of oocytes from AIS-hCG females decreased compared to PMSG-hCG (P<0.001). The PVS of oocytes from the AIS-hCG was larger than PMSG-hCG (P<0.001). The microtubules of oocytes from both AIS-hCG and PMSG-hCG were normal although there was an increased fluorescence intensity in the AIS-hCG oocytes (P<0.001). The F-actin and CGs distribution of oocytes from both AIS-hCG and PMSG-hCG were similar (P=0.330 and P=0.13, respectively). While the oocytes from PMSG-hCG females had homogenously distributed mitochondria, AIS-hCG showed more peripheral distribution with no differences in fluorescence intensity (P=0.137). The blastocyst development rates after IVF with fresh sperm showed no difference between AIS-hCG and PMSG-hCG (P=0.235). These data suggest that AIS-hCG superovulation produced high numbers of morphologically normal oocytes and also possessed normal subcellular structures, good morphological characteristics and had high in-vitro embryonic developmental potential.

Summary

The gonadotropic hormones used for superovulation may influence the quantity and quality of resulting mouse oocytes and embryo development following IVF and embryo culture. We evaluated quantitative, morphometric, subcellular characteristics, in-vitro fertilization and embryonic developmental competence of the oocytes derived after anti-inhibin serum (AIS) or PMSG administration. This information would be useful for those who are interested in mouse oocyte and embryo development, genetic modifications and assisted reproductive technologies in mice.

Introduction

The production of a sufficient quantity of good quality oocytes is very critical for biomedical research and assisted reproductive technologies (ARTs) such as in-vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), transgenic animal production, somatic cell nuclear transfer (Behringer et al., 2014) and germplasm cryo-banking (Agca, 2012). Therefore, the availability of effective superovulation procedure is very important for producing an adequate number of oocytes and reducing the number of donor females particularly for genetically modified rodents and strains showing poor response to superovulation procedure (Hasegawa et al., 2016; Takeo and Nakagata, 2016).

Pregnant mare’s serum gonadotropin (PMSG) is a unique gonadotropin among mammals and most widely used for superovulation of laboratory mice (Behringer et al., 2014). PMSG functions similar to follicle stimulating hormone (FSH) (Behringer et al., 2018) which selects and stimulates multiple ovarian follicles to grow and mature by forming functional complexes with both FSH and luteinizing hormone (LH) receptors. Inhibins are glycoprotein hormones composed of two molecular forms which are inhibin A and inhibin B (Muttukrishna, 2004). They are mainly produced from ovarian follicles and they inhibit the production of the FSH from the anterior pituitary gland (Medan et al., 2007). Inactivation of inhibin is a very effective way for superovulation of various mammalian species including mice (Wang et al., 2001), rats (Ishigame et al., 2004), hamsters (Kishi et al., 1996), guinea pigs (Shi et al., 2000), cows (Takedomi et al., 1997), mares (Nambo et al., 1998), ewes (Medan et al., 2007) and goats (Medan et al., 2003). Using the AIS to immune-neutralize inhibin in cycling rats increased the production of FSH (Gordon et al., 2010). Similarly, injection of female mice with AIS caused an increase in endogenous production of FSH that stimulated follicular growth in the ovaries (Hasegawa et al., 2016). Use of AIS is a very effective way to produce adequate numbers of oocytes from genetically modified inbred and outbred mouse strains (Takeo and Nakagata, 2016).

Oocyte quality is one of the most important factors for successful fertilization and embryo development, both of which are critical for successful pregnancy. Thus, examining and selecting the morphological normal, good quality oocytes is very important for successful execution of ART. The integrity of the microtubules is one of the most important determining factors for the quality of the oocyte. Microtubules play crucial roles during the course of chromosome alignment at Metaphase II and correct segregation of the sister chromatids during anaphase II (Vogt et al., 2008; Tomari et al., 2018). Errors in chromosome segregation can cause aneuploidy which may result in spontaneous abortions and pregnancy loss in mice (Akiyama et al., 2006; Selesniemi et al., 2011) and humans (Bond and Chandley, 1983).

Similarly, F-actin is an important cytoskeletal component that contributes to maintenance of oocyte shape, migration of meiotic spindles and chromosomes, cytoplasm, polar body extrusion, spindle positioning, fertilization and cell division during embryo development. In addition, F-actin is important for the distribution, movement and exocytosis of cortical granules (CGs) during oocyte maturation and fertilization (Kim et al., 1996). The altered distribution of F-actin may cause decreased IVF, lower cleavage and reduced pre-implantation embryo development rate (Ci et al., 2014). The CGs are the Golgi derivative membrane bound regulatory secretory organelles that range from 0.2 μm to 0.6 μm in diameter and are found in the cortex of unfertilized MII oocytes of most species (Cran and Esper, 1990; Liu, 2011). During fertilization, an increased influx of Ca²⁺ causes the single membrane of the CGs to merge with the oocyte membrane and release their contents into the perivitelline space and changes the structure of zona pellucida (ZP). These cascades of events ultimately block the polyspermic fertilization (Ghetler et al., 2006). The mitochondria are considered as one of the important indicators of oocyte quality and influence successful fertilization and embryonic development (Schatten et al., 2014). Mitochondria contain genetic materials that are transferred from the oocytes to the offspring and their dysfunctions have been implicated with many inherited disorders including diabetes and deafness (Murphy et al., 2008), Leigh’s Disease (Murphy and Craig, 1975), Neuropathy, ataxia, and retinitis pigmentosa syndrome (Thorburn et al., 1993) in the offspring.

The ZP is an oocyte extracellular matrix that is composed of at least three or four glycoproteins named ZP1, ZP2, ZP3 and ZP4 (Stetson et al., 2015; Moros-Nicolas et al., 2017). They collectively play an important role during the fertilization, embryo development and implantation. The ZP thickness of human oocytes was correlated to fertility since oocytes having relatively thinner ZP resulted in a higher IVF rates, while thicker ones often required ICSI (Bertrand et al., 1995). Similarly, rabbit oocytes having thicker ZP also caused failed fertilization compared to oocyte with thinner ZP (Marco-Jimenez et al., 2012).

The perivitelline space (PVS) is the cavity between the oocyte plasma membrane and the ZP, and is rich in extra-cellular matrix components which are essential for fertilization, implantation and embryo development. For humans, screening of large numbers (1,622) of MII human oocytes from 160 ovarian stimulation cycles and 126 oocyte donors (Ten et al., 2007) revealed that oocyte PVS larger than normal have a 1.8 times higher chance to develop into good quality of embryos.

To our best knowledge, this is one of the first comprehensive studies which compared the mouse oocyte diameter, zona pellucida thickness, perivitelline space, and subcellular structures (microtubules, F-actin, cortical granules, mitochondria) derived from AIS-hCG or PMSG- hCG superovulation methods. In addition, IVF and embryo culture were performed on AIS-hCG and PMSG-hCG derived oocytes using either fresh or frozen-thaw sperm to determine fertility and developmental competence to blastocyst stage.

Materials and Methods

Animals

In this study, 8-12 week old outbred ICR mice and C57BL/6 inbred mice were used (n= 58, Charles River Wilmington, MA). All of the animal studies were approved by the University of Missouri’s Animal Care and Use Committee and were in accordance with the guidelines of the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals. On arrival, the mice were housed in micro-isolator caging at a temperature range of 20-25°C and lighting (10-h dark/14 h light) controlled environment and provided free access to water and standard pelleted rodent chow. Animals were checked daily by the animal care staff.

Chemicals

All chemicals used in this experiment were obtained from Sigma Chemical Company (St. Louis, MO) or ThermoFisher Scientific unless stated otherwise.

Superovulation and oocyte collection

Each mouse was injected intraperitoneally with 7.5 IU of PMSG or 100 μl of AIS (CARD institute, Japan) followed by the injection of 7.5 IU hCG 48 post AIS or PMSG. The AIS had a titer of 1:1000000 as defined by the final dilution of the antiserum required to bind 50% of ¹²⁵I-labeled bovine 32-kDa inhibin. The clutches of cumulus-oocyte complexes (COCs) were collected from the oviducts 14-15 hours post hCG injection.

Sperm collection and freezing

The cauda epididymides were dissected out and placed in 1.2 mL 18% raffinose pentahydrate (w/v) and 3% skimmed milk (w/v) freezing solution as previously described (Takeo and Nakagata, 2011). A small cut was made by using sterile micro-scissors by securing each epididymis with a fine tweezer. After 10 min the freezing solution containing the sperm were loaded into 0.25 mL French straws and placed in a Styrofoam box containing LN2. The straws containing the sperm were then cooled in vapor phase of LN2 on a mash wire above LN2 for 5 min, and then plunged into LN2 for long term storage.

In Vitro Fertilization (IVF) and embryo culture

For IVF with frozen-thawed sperm, the straws containing frozen sperm were removed from LN2 and thawed in a water bath at 37°C. The content of the straw was gently expelled on top of 1 mL FHM media containing BSA (4 mg/mL) in an Eppendorf tube and then centrifuged for 5 min at 300 g. The supernatant was removed and the sperm pellet was gently pipetted out and placed into a pre-equilibrated 95 μL FERTIUP drop for sperm capacitation. For IVF with fresh sperm, an adult proven male mouse was euthanized by cervical dislocation and the cauda epididymides were gently dissected out and cleaned of any blood and fat on a clean tissue paper and placed in mineral oil next to the FERTIUP sperm pre-incubation drop. With the aid of a fine tweezers a small cut was made in each epididymis. A sterile 27g needle was used to squeeze out dense sperm and immediately pulled into a 90 μL drop of FERTIUP for sperm capacitation for 30 min in an atmosphere of 5% CO2, in air at 37°C before IVF procedure.

IVF procedure was performed as previously described (Nakagata, 2011; Takeo et al., 2008). Both FERTIUP and CARD (for in-vitro fertilization) media were pre-equilibrated in a humidified incubator containing 5% CO₂ in air under mineral oil at 37°C in 35 mm Petri dishes (Falcon 351008). For IVF, previously dissected oviducts from each donor female were separately placed into mineral oil next to a CARD drop and under a stereomicroscope. The clutches of COCs from each donor were first released into the mineral oil with the aid of fine tweezers and 28 g needle and then gently pulled into 90 μL CARD drops. For IVF, frozen-thawed sperm were capacitated in FERTIUP and then gently added (~3.5x10⁵ motile sperm/mL) into each CARD drop containing the clutches of COCs. After about 6 hours of sperm and egg co-incubation, the presumptive zygotes were washed 3 times in FHM media containing BSA (4 mg/mL) and transferred into KSOM amino acid culture drops (Ho et al., 1995; Summers, 2013) for determination of two-cell, morula and blastocyst development.

Immunofluorescence staining and Confocal Microscopy

For fluorescence staining, oocytes from both AIS and PMSG cumulus oocyte complexes were dispersed by using 1 mg/ml hyaluronidase to remove the cumulus cells and 5 mg/ml protease to remove the ZP. Denuded oocytes were then fixed with 3.7% formaldehyde for 30 min and then permeabilized with 0.3% polyvinylpyrrolidone (PVP) + 0.1% Tween-20 + 0.01% Triton X-100 for 20 min. This was followed by blocking in 1% BSA in PBS containing 0.1% Triton for 1 hour. Then, the oocytes in each group were incubated with mouse monoclonal anti-α-tubulin–FITC antibody (clone DM1A, purified from hybridoma cell culture from Sigma, Catalog number: F2168; diluted 1:200 with blocking buffer) for 60 min at room temperature. For F-actin staining, the oocytes in each group were incubated in 1μg/ml of phalloidin-TRITC (Rhodamine Phalloidin, Invitrogen, catalog number # R415) at room temperature for 30 min. For cortical granules staining, the oocytes in each group were stained with LCA-FITC (Lectin from Arachis hypogaea conjugated with FITC, Sigma, Catalog number: L7381; 1:100 dilution with PBS) for 1 hour at room temperature. After three washes in PBS containing 0.3% PVP, the oocytes were transferred into a small drop of prolong Antifade mounting medium containing 4, 6 –diamidino-2-phenylindole (DAPI) on a microscope slide and covered with a coverslip.

Confocal laser-scanning images were obtained by Leica Confocal (Leica, Germany). For mitochondria staining, the oocytes in each group were incubated with rabbit polyclonal customized phosphate carrier primary antibody overnight, and then stained with goat anti-rabbit IgG secondary antibody, Alexa Flour plus 647 (Invitrogen) at a dilution of 1:1000 for 1hr at RT. After three washes in PBS containing 0.3% PVP, the oocytes were transferred into a small drop of prolong Antifade mounting medium containing DAPI on a microscope slide and covered with a coverslip. Confocal laser-scanning images were obtained by using Leica Confocal (Leica, Germany).

Statistical analysis

All IVF, embryonic development, ZP thickness, perivitelline space, oocyte diameter, F-actin, microtubules, CGs, and mitochondria fluorescence intensity data from AIS-hCG and PMSG-hCG superovulation groups were analyzed by t-test comparison by using SigmaPlot 14.0 (Systat Software Inc.) and graphs were created based on the analyzed results.

Results

A total of 744 oocytes were collected from 14 donors after AIS-hCG and the total number of 500 oocytes were collected from 17 donors after PMSG-hCG. The mean number of the oocytes per female obtained from AIS-hCG (n=53.3) was higher than the PMSG-hCG (n=29.4, P<0.001; Figure 1). Morphologically normal oocytes from AIS-hCG and PMSG-hCG methods were 90.2% (671/744) and 87.4% (437/500), respectively. The average number of morphologically normal oocytes per donor with the AIS-hCG (n=47.9) was higher than PMSG-hCG (n=25.7, P<0.001; Figure 2, A and B). There was no significant difference in the average number of the morphologically abnormal (lysed, fragmented) oocytes between AIS-hCG (n=4.9) and PMSG-hCG (n=4.2, P=0.425; Figure 2, C and D).

Figure 1.

Average number of MII mouse oocytes obtained from each donor female after either AIS-hCG or PMSG-hCG superovulation methods.

Figure 2.

The average number (A) and percent (B) of morphologically normal MII mouse oocytes from each donor female after either AIS-hCG or PMSG-hCG superovulation methods. The average number (C) and percent (D) of morphologically abnormal MII mouse oocytes from each donor female after either AIS-hCG or PMSG-hCG superovulation methods.

In addition to gross morphological evaluation, morphometric analysis were performed to determine the diameter, zona pellucida (ZP) thickness and perivitelline space (PVS) of the oocyte derived from AIS-hCG or PMSG-hCG stimulation. The ZP thickness of oocytes collected from AIS (7.92± 0.075 μm) was thinner (8.74±0.083 μm) than those ZP of PMSG-hCG derived oocytes (P<0.001; Table 1). The PVS of oocytes derived from AIS-hCG (10.98 ± 0.439 μm) was larger than those obtained from the PMSG-hCG (7.16± 0.347 μm) method (P<0.001; Table 1). There was no statistical difference in oocyte diameter between AIS-hCG (71.17 ± 0.28 μm) PMSG-hCG (73.15 ± 0.30 μm, P=0.289; Table 1).

Table 1.

The average diameter, PVS, and thickness of the ZP of MII mouse oocytes derived from either AIS-hCG or PMSG-hCG superovulation methods.

Treatments	No. oocytes	Oocyte diameter	Perivitelline space	Thickness of ZP
PMSG-hCG	100	73.15 ±0.30a μm	7.16± 0.347a μm	8.74±0.083a μm
AIS-hCG	100	71.17 ±0.28a μm	10.98 ±0.439b μm	7.92± 0.075b μm

Note: Values with different letters within a column are different (p < 0.05).

Further investigation was performed to determine if AIS-hCG and PMSG-hCG stimulation produces oocytes with similar subcellular organelle properties. Although the oocytes from both AIS-hCG (n=47) and PMSG-hCG (n=100) maintained normal microtubule, structural integrity, AIS derived oocytes had significantly higher fluorescence intensity than PMSG-hCG (P<0.001; Figures 3A-C). The F-actin in both groups had normal distribution and there was no difference in fluorescence intensity between AIS-hCG or PMSG-hCG (P=0.33; Figures 3D-F).

Figure 3.

The representative confocal images of microtubules (A, B) and corresponding fluorescence intensity measurement (C) of MII oocyte derived from either AIS-hCG or PMSG-hCG, respectively; microtubules (green) with DNA (blue). The representative confocal images of F-actin (D, E) and corresponding fluorescence intensity measurement (F) of MII mouse oocytes derived from either AIS-hCG or PMSG-hCG superovulation method, respectively.

Both AIS-hCG and PMSG-hCG derived oocytes had normal CGs distribution with no significant difference in fluorescence intensity (P=0.13; Figures 4A-C). While the oocytes obtained from PMSG-hCG had more homogenously distributed mitochondria, AIS-hCG derived oocytes showed a more peripheral distribution within the cytoplasm (Figures 4D and E). Fluorescence intensity of mitochondrial staining were similar between AIS-hCG and PMSG-hCG derived oocytes (P=0.137; Figure 4F).

Figure 4.

The representative confocal images of cortical granules (A, B) and corresponding fluorescence intensity measurement (C) of MII oocyte from either AIS-hCG and PMSG-hCG superovulation methods, respectively. The representative confocal images of mitochondria distribution (D, E) and corresponding fluorescence intensity measurement (F) of MII oocyte from either AIS-hCG and PMSG-hCG superovulation methods, respectively.

There were no difference in 2-cell embryo development between AIS-hCG (86.2%) and PMSG-hCG (75.9%) stimulated oocytes (P=0.103; Figure 5A). There was also no significant difference in morula formation rates (94.3% vs 90%, P=0.235; Figure 5B) and blastocyst formation rates (96.8% vs 94.7%, P=0.203; Figure 5C) between AIS-hCG and PMSG-hCG, respectively. Although there was 10.6% higher 2-cell embryo development for the AIS-hCG derived oocytes (71.5%) compared to PMSG-hCG (60.9%), this was not statistically significant (P=0.189; Figure 5D). However, there was a significantly higher morula (91.0% vs 86.9%, P=0.01; Figure 5E) and blastocysts formation (95.4% vs 89.9%, P=0.032); Figure 5F for AIS-hCG compared to the PMSG-hCG superovulation methods, respectively.

Figure 5.

Two-cell (A), morula (B) and blastocyst (C) development of either AIS-hCG or PMSG-hCG derived MII oocytes following in-vitro fertilization with fresh sperm and subsequent in-vitro embryo culture. Two-cell (D), morula (E) and blastocyst (F) development of either AIS-hCG or PMSG-hCG derived MII oocytes following in-vitro fertilization with frozen-thawed sperm and subsequent in-vitro embryo culture.

Discussion

Several previous studies compared the superovulation efficiencies between AIS-hCG and PMSG-hCG protocols. It has been reported that passive immunization of inhibin through AIS-hCG produces 1.5 to 3.2 times more oocytes than PMSG-hCG in commonly used inbred (C57BL/6, A/J, BALB/cByJ, C3HeJ, DBA/2J, FVB/NJ, BALB/cA), outbred (ICR) and hybrid (B6D2F1) mouse strains as well as Japanese wild-derived Mus musculus molossinus (Hasegawa et al., 2012; Takeo and Nakagata, 2016). It has been further suggested that superovulation by AIS-hCG produces similar quality mouse oocytes, IVF embryos and apparently healthy live offspring compared to PMSG-hCG (Hasegawa et al., 2016). However, given the large numbers of oocytes obtained from AIS-hCG, we hypothesized that there may be potential changes in morphometric, subcellular and embryo development characteristics of oocytes obtained from AIS-hCG. One of the striking findings in this study was that ZP of oocytes derived from AIS-hCG had significantly thinner ZP than those oocytes derived from PMSG-hCG. This may indicate a higher potential for IVF success according to previous studies on human and rabbit oocytes. For Mil human oocytes, the thickness of the ZP was linked to IVF success (Bertrand et al., 1995). A relatively thinner (16.6 μm) ZP resulted in higher IVF rate and the ZP thickness equal to or thicker than 22 μm was needed ICSI. Similarly, in rabbits, oocytes with thicker ZP (19.2 μm) caused failed fertilization while the oocytes having thinner ZP (18.5 μm) resulted in successful fertilization (Marco-Jimenez et al., 2012). On the other hand, Hasegawa et al., (2016) did not find significant difference in ZP digestion time between AIS-hCG and PMSG-hCG derived oocytes after exposing C57BL/6 oocytes to 1% α-chymotrypsin.

In this study, the PVS of the oocytes produced from the AIS-hCG were larger than the PVS of oocytes induced from the PMSG-hCG method. Larger PVS was shown to improve embryo quality in humans (Ten et al., 2007). The cytoplasmic volume is an important determinant for oocyte quality since it is the indicative for the amount of organelles and amount of metabolites that influence overall oocyte quality (Reader et al., 2017; Liu et al., 2018). In this study, the oocyte diameters derived from AIS-hCG or PMSG-hCG were not different. Thus, based on the cytoplasmic volume, the current study suggests that both AIS-hCG and PMSG-hCG methods yield equal quality oocytes.

The microtubules of oocytes derived from both superovulation methods had good integrity, but AIS-hCG derived oocytes showed significantly higher fluorescence intensity. This suggests greater amount of microtubules in oocytes produced from AIS-hCG treatment and improved in-vitro developmental potential of the embryos since integrity of the microtubules plays an important role for alignment of chromosomes in the MII stage oocytes and correct segregation of the sister chromatids during anaphase II (Vogt et al., 2008; Tomari et al., 2018). Actin filaments within the developing and fully mature oocytes are responsible for many crucial events including maintaining morphology, migration of meiotic spindles and chromosomes positioning, polar body extrusion, and cell division (Lenart et al., 2005; Azoury et al., 2009). Distribution of F-actin was similar in both AIS-hCG and PMSG-hCG derived oocytes and there was no significant difference in fluorescence intensity. This indicates quality of the oocytes from both methods were comparable.

CGs play an important role in blocking polyspermy and embryonic development for ensuring normal fertilization and development of the oocytes (Wessel et al., 2001; Ghetler et al., 2006). The CGs integrity of both AIS-hCG and PMSG-hCG derived oocytes were comparable and no difference in fluorescence intensity was detected. This finding further demonstrates the production of similar quality of oocytes in both AIS-hCG and PMSG-hCG groups.

Mitochondrial viability and their distribution within the oocyte is a dynamic process and may provide important information with regards to the oocyte quality and healthy embryonic development (Schatten et al., 2014). In this study, the mitochondria were more homogenously distributed in the oocytes derived from PMSG-hCG whereas more peripherally distributed in the oocytes derived from AIS-hCG. However, the fluorescence intensity of mitochondria in two methods were not statistically different which provides further evidence for production of good quality of mouse oocytes in both superovulation methods.

The C57BL/6 are the most commonly used inbred strain for transgenic mouse production and these strains are often re-derived via IVF using fresh or frozen sperm. Thus, it is crucial to investigate the quantity (oocyte yield), quality (fragmentation and lysis), IVF and in-vitro developmental potential of the oocytes derived from AIS-hCG or PMSG-hCG using fresh or frozen sperm. The mean number of the oocytes from each donor in the AIS-hCG method was significantly higher than the mean number of the oocytes ovulated from each clutch in the PMSG-hCG superovulation group. The current study is consistent with the results of the previous studies which also found significantly increased oocyte yield from AIS-hCG in inbred C57BL/6 strain without compromising their morphological integrity (Hasegawa et al., 2012; Hasegawa et al., 2016).

We also performed IVF and in-vitro embryo culture to determine if the morphometric and subcellular characteristics of the oocytes obtained from AIS-hCG or PMSG-hCG correlate with fertility and embryonic development competence. The IVF and subsequent embryo development competence to blastocyst stage with the use of either fresh or frozen thawed sperm showed a similar trend. Despite a slight increase in the 2-cell embryo development for oocytes obtained from AIS-hCG than PMSG-hCG, this difference was not significant for either fresh or frozen-thawed sperm. The slight increase in 2-cell development rates for AIS-hCG oocytes with the use of either fresh or frozen-thawed sperm may be due to higher sperm penetration rates of AIS-hCG derived oocytes with thinner ZP. Similar to 2-cell development, the in-vitro blastocyst formation rates of 2-cell embryos produced via IVF by using fresh sperm were not different between AIS-hCG and PMSG-hCG. However, higher rate of morula and blastocysts formation were detected for AIS-hCG than the PMSG-hCG when frozen thaw sperm was used for IVF. Overall, embryo development rates in this study with either fresh or frozen-thawed sperm was consistent with the results from a previous studies which compared the AIS-hCG and PMSG- hCG methods for C57BL/6 mouse strain (Hasegawa et al., 2012, Takeo and Nakagata, 2015, Hasegawa et al., 2016; Takeo and Nakagata, 2016).

In conclusion, the current study collectively suggests that the AIS-hCG method is an extremely effective superovulation method for the production of high quality mouse oocytes having comparable subcellular structural characteristics with PMSG-hCG in terms of microtubules, F-actin, cortical granules, and mitochondria. Furthermore, IVF with fresh or frozen thawed sperm as well as in-vitro embryonic development competence were also well correlated with the quality of subcellular properties between AIS-hCG and PMSG-hCG superovulation methods. Therefore, AIS can be a very effective reagent for the superovulation of particularly low-responding mouse strains for biomedical research.