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Superovulation and in vitro oocyte maturation in three species of mice (Mus musculus, Mus spretus and Mus spicilegus)
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Theriogenology 70 (2008) 1004–1013 www.theriojournal.com
Superovulation and in vitro oocyte maturation in three species of mice (Mus musculus, Mus spretus and Mus spicilegus) J. Martı´n-Coello a, R. Gonza´lez a, C. Crespo a, M. Gomendio a, E.R.S. Roldan a,b,* a
Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC), c/Jose´ Gutie´rrez Abascal 2, 28006 Madrid, Spain b Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 0TU, UK Received 17 April 2008; received in revised form 5 June 2008; accepted 8 June 2008
Abstract Mouse oocytes can be obtained via superovulation or using in vitro maturation although several factors, including genetic background, may affect response. Our previous studies have identified various mouse species as models to understand the role of sexual selection on the evolution of sperm traits and function. In order to do comparative studies of sperm–oocyte interaction, we sought reliable methods for oocyte superovulation and in vitro maturation in mature females of three mouse species (genus Mus). When 5 IU pregnant mare’s serum gonadotrophin (PMSG) and 5 IU human chorionic gonadotrophin (hCG) were injected 48 h apart, and oocytes collected 14 h post-hCG, good responses were obtained in Mus musculus (18 1.3 oocytes/female; mean S.E.M.) and Mus spretus (12 0.8), but no ovulation was seen in Mus spicilegus. Changes in PMSG or hCG doses, or longer post-hCG intervals, did not improve results. Use of PMSG/luteinizing hormone (LH) resulted in good responses in M. musculus (19 1.2) and M. spretus (12 1.1) but not in M. spicilegus (5 0.9) with ovulation not increasing with higher LH doses. Follicular puncture 48 h after PMSG followed by in vitro maturation led to a high oocyte yield in the three species (M. musculus, 23 0.9; M. spretus, 17 1.1; M. spicilegus, 10 0.9) with a consistently high maturation rates. In vitro fertilization of both superovulated and in vitro matured oocytes resulted in a high proportion of fertilization (range: 83–87%) in the three species. Thus, in vitro maturation led to high yields in all three species. These results will allow future studies on gamete interaction in these closely related species and the role of sexual selection in gamete compatibility. # 2008 Elsevier Inc. All rights reserved. Keywords: Superovulation; In vitro maturation; Oocyte; Sperm; Mouse
1. Introduction A reliable supply of large numbers of high quality oocytes, with a good competence for fertilization and development, is an important requisite both for basic reproductive studies and for applied research on assisted
* Corresponding author at: Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC), c/Jose´ Gutie´rrez Abascal 2, 28006 Madrid, Spain. Tel.: +34 91 411 13 28x1245; fax: +34 91 564 50 78. E-mail address: [email protected] (E.R.S. Roldan). 0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.06.002
reproductive techniques. Basic studies require a good oocyte supply to investigate female gamete biology and sperm–egg interactions, providing valuable information about the underlying physiological and cellular mechanisms, as well as about the evolution of species-specificity and reproductive barriers. In the context of assisted reproduction, oocyte supply is crucial for the preservation of genetic resources, conservation of endangered species, transgenesis, or cloning by nuclear transfer, either for reproductive purposes or for the generation of embryonic stem cells to be used in regenerative medicine. Thus, the ability to collect as many unfertilized eggs as possible, and at any time of the day, is an essential tool
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needed for experimental animals, domestic species and endangered wildlife. There are standardized procedures for oocyte superovulation and collection in the laboratory mouse [1], but these may not be applicable to other species. Interest on studies of comparative gamete biology has led to the use of wild mouse species introduced to laboratory conditions [2] and this, in turn, has generated the need to develop new techniques of oocyte collection. Oocyte growth occurs primarily in preantral follicles where the oocyte is arrested in prophase I of meiosis. In a subsequent stage, follicles are selected under the action of follicle stimulating hormone (FSH) and they undergo an increase in size and specialization of granulosa cells into cumulus and mural granulosa cells. The final stage of follicle maturation involves the period between luteinizing hormone (LH) surge and ovulation, and during this time, the oocytes resume meiosis (which arrests at metaphase II) and complete their cytoplasmic maturation; parallel processes in the follicle involve cumulus oophorus expansion, follicle luteinization, and breakdown of the follicle basal lamina [3–5]. These processes can be stimulated with exogenous gonadotrophins and can be supported, in part, during in vitro culture. In mice, about 8–12 ova are released at ovulation, depending on the strain. These numbers can be substantially increased by using exogenous hormones, thereby reducing the animals needed for experimentation [1]. Two main approaches can be used to obtain mature oocytes, namely superovulation (i.e., maturation in vivo) and maturation in vitro. Superovulation involves hormonal treatments to induce follicular development and oocyte release [1,6,7]. Hormones used to induce follicular development are FSH, pregnant mare’s serum gonadotrophin (PMSG) or human menopausal gonadotrophin (HMG). Hormones used to induce follicular rupture and oocyte release include LH and human chorionic gonadotrophin (hCG). Alternatively, hormones such as fertirelin acetate, an LH-releasing hormone analogue, have been used to successfully induce superovulation in mice [8,9]. Induction of superovulation in this species has also relied on the administration of inhibin antiserum [10]. Superovulation with a PMSG/hCG combination is known to induce ovulation in the mouse at any stage to the oestrous cycle [11,12]. This hormonal treatment has also been used to induce superovulation in various rodent species including rats [13,14], Syrian (golden) hamster [15], Siberian (Djungarian) hamster [16] and vesper mice [17,18], but it may sometimes be inefficient in some species such as the Chinese hamster [19]. On
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the other hand, a PMSG/LH combination has been used to reliably induce superovulation in several species [19– 21]. Although superovulation is generally successful for rodent species, it has been found that different strains do not respond in the same way to hormonal treatment (mice: [22,23]; rat: [24,25]). Since superovulation treatments do not increase levels of endogenous gonadotrophins [26], variation in ovarian response to exogenous hormones is likely due to genetic differences between strains [27,28]. Therefore, in order to obtain similar high responses among strains (or species), it may be necessary to adjust hormone concentrations to account for these differences in ovarian response. The response to superovulation treatments varies with age and weight, nutritional status, health and housing conditions of females [1,6]. There are variations with age in the number of ovulated ova per female, with younger females ovulating more oocytes than older ones [6,29–31]. Similarly, females with growth delay, as evidence by lower body weight, tend to yield reduced numbers of oocytes [32]. The dose of administered gonadotrophins [30], the potency [33] and the timing of administration can affect the sensibility to the superovulation treatment and may also be of critical importance for embryo production and development of embryos to the blastocyst stage. Superovulatory response (and subsequent embryo development) can also vary depending on the day of the oestrus cycle when PMSG is administered [34,35]. An alternative approach used to obtain a high number of oocytes involves in vitro maturation (IVM). Studies have established that IVM of oocytes from the germinal vesicle stage to the metaphase II (MII) stage is technically feasible in a range of different mammalian species [20,36], including laboratory animals such as mice and rats [37–39]. The protocol can include an initial administration of hormones such as PMSG or FSH in order to induce development of a higher number of follicles, followed by the maturation of cumulus– oocytes complexes (COCs) obtained from antral follicles in culture medium. In previous work, we have characterized sperm traits and function in various species of mice [40]. In order to test fertilizing capacity of spermatozoa from these wild mice, it is necessary to collect oocytes either via superovulation or in vitro maturation. Preliminary work using standard superovulation protocols for the laboratory mouse revealed considerable differences in response between two species and the inability to obtain a superovulatory response in another one. This highlighted the need to test modifications of this protocol, and to search for alternative means to obtain
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mature oocytes. The objective of this study was to explore conditions for superovulation and in vitro oocyte maturation that would lead to the collection of as many viable mature oocytes as possible in three species of mice from the genus Mus (Mus musculus, Mus spretus and Mus spicilegus) and to evaluate the ability of these oocytes to undergo fertilization in vitro. 2. Materials and methods 2.1. Animals All animal procedures were performed following the Spanish Animal Protection Regulation, RD1201/2005 which conforms to European Union Regulation 2003/ 65. Procedures also adhered to the Guiding Principles for Biomedical Research Involving Animals, as issued by the Council for the International Organizations of Medical Sciences and recommended by the journal. Adult females and males of three species of Murid rodents (M. musculus, M. spicilegus and M. spretus) were purchased from the Laboratoire Ge´nome et Populations, Universite´ de Montpellier II, and kept using standard laboratory mouse conditions, in an environmentally controlled room on a 14 h light:10 h darkness photoperiod under constant temperature and relative humidity conditions. Under these conditions, animals reproduce throughout the year. Animals were provided with food (Harlan Ibe´rica, Barcelona, Spain) and water, both available ad libitum. Animals to be used for experiments were weaned when they were 4 weeks old. Males were kept in individual cages and were used when they were >16 weeks old. After weaning, females were housed together and they were used when they were 12–20 weeks old. We chose to use females of these ages because these wild-derived animals reach maturity at about 3–4 months of age. In addition, we decided against using immature animals for superovulation because one of the aims of future work involves mating or artificial insemination of superovulated animals; immature animals would not be adequate for this type of experimental work. Species used in this study differ in the number of pups per litter (mean S.E.M): M. musculus, 6.9 0.4; M. spretus, 5.7 0.4 and M. spicilegus 3.7 0.5. 2.2. Superovulation Females of three species of mice were subjected to two different hormonal treatments to induce superovulation. In the first hormonal treatment females were injected intraperitoneally with 5 IU of PMSG (Folligon,
Intervet, Madrid, Spain) and 5 IU of hCG (Chorulon, Intervet, Madrid, Spain) 48 h later. Oocytes were recovered 14 h post-hCG. This is the standard superovulation treatment for CD1 and B6D2F1 mice used in our laboratory, as well as by other authors [22,23,41,42]. Due to a limited response in M. spicilegus further studies were carried out to induce superovulation in this species. Females were injected with 2.5 IU of PMSG and 2.5 IU of hCG, or with 7.5 IU of PMSG and 7.5 IU of hCG; in both cases oocyte recovery was performed 14 h posthCG. The effect of time of oocyte recovery after hCG injection was also examined in M. spicilegus females given 5 IU of PMSG and 5 IU of hCG. In the second hormonal treatment, superovulation was induced with PMSG and LH (Sigma, Madrid, Spain) based on results in other species [19,43]. Females of the three species were intraperitoneally injected with 5 IU of PMSG followed 48 h later by 400 IU of LH and oocyte collection 14 h post-LH. In addition, superovulation of M. spicilegus females was attempted with 5 IU of PMSG and different LH concentration (250 IU, 300 IU, 350 IU, 400 IU and 450 IU). After hormonal treatments females were sacrificed by cervical dislocation, reproductive tracts were removed and placed in human tubular fluid (HTF) [44]. Oocyte-containing cumulus masses were released from the ampullae with the aid of a pair of needles under a dissecting microscope and used for in vitro fertilization. 2.3. Oocyte collection from antral follicles and in vitro maturation Immature mouse oocytes were collected from females of the three species 48 h after administration of an intraperitoneal injection of 5 IU of PMSG. In vitro maturation of oocytes was performed as described previously [45]. Ovaries were dissected out and placed in Petri dishes containing M2 medium (Sigma) supplemented with 5% (v/v) heat-inactivated foetal bovine serum (FBS, Gibco, Invitrogen, Barcelona, Spain). COCs were released by follicular puncturing with the aid of a pair of 30 G needles attached to disposable syringes under a stereomicroscope. Only immature oocytes surrounded by compact cumulus cells were selected. After several washes in M2, groups of 20–50 COCs were placed in 4-well culture dishes (Nunclon, Nalgene, Nunc International, Roskilde, Denmark) containing the in vitro maturation medium, covered with 250–300 ml of mineral oil and cultured at 37 8C in 5% CO2 in air and maximum humidity for
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17 h. A volume of 500 ml of minimum essential medium-alpha supplemented with Earle’s salts (MEM-alpha, Gibco, Invitrogen) plus 10 mg/ml streptomycin sulfate, 75 mg/ml penicillin G (Sigma) and 5% (v/v) heat-inactivated FBS was used as maturation medium. Meiosis progression until metaphase II (MII) was confirmed by the presence of the polar body after the incubation period. Maturation rate was recorded and expressed over the total number of oocytes cultured. Mature oocytes were used for in vitro fertilization. 2.4. In vitro fertilization Mouse epididymides were removed from males of the three mouse species and placed in separate 1 ml drops of HTF medium under oil, and incubated at 37 8C in 5% CO2/air for 10 min to allow for sperm dispersion. After incubation, epididymal tissue was discarded and the sperm suspensions were incubated at 37 8C under 5% CO2 in air for capacitation as described before [40]. In vitro fertilization was carried out in 500 ml droplets of HTF medium. Oocytes from each species, obtained after superovulation or after in vitro maturation, were placed in HTF medium. Each sample was inseminated with a final concentration of 0.8–1 106 sperm/ml of capacitated spermatozoa. Gametes were co-incubated for 4 h at 37 8C under 5% CO2 in air. After this period, remaining cumulus cells and attached sperm were removed by washing the oocytes in and out with a fine pipette. Oocytes were then washed three times with HTF medium and placed in 150 ml HTF drops. Fertilization was assessed by pronucleus observation under microscope examination. Fertilization rate was expressed over the number of superovulated or in vitro mature oocytes that were co-incubated with spermatozoa. 2.5. Statistical analysis Variables were transformed to fulfill parametric assumptions: percentages were arcsine-transformed whereas all other variables were log-transformed. Differences between treatments were tested using ANOVA and Tukey tests. A value of p < 0.05 was regarded as indicative of statistical significance. 3. Results 3.1. Superovulation When females were treated with 5 IU of PMSG and 5 IU of hCG, and oocytes recovered 14 h post-hCG, an average of 18 oocytes/female were collected from M.
Fig. 1. Superovulation in three mouse species (Mus musculus, M. spretus and M. spicilegus) after treatment with PMSG and hCG. Females of each species were treated with 5 IU of PMSG followed, 48 h later, by 5 IU of hCG. Oocytes were collected from oviducts 14 h after hCG administration. Number of females used for each treatment is shown in parenthesis. Values are means S.E.M. Differences in the number of oocytes recovered from M. musculus and M. spretus were statistically significant ( p < 0.05).
musculus, and 12 oocytes/female from M. spretus (Fig. 1); on the other hand, oocytes were not recovered from M. spicilegus although there were mature follicles in the ovaries. The proportion of abnormal oocytes observed was low in the species that reponded to superovulation, being on average 1.4 (7.7%) abnormal oocytes per female in M. musculus and 1.3 (15.9%) abnormal oocytes per female in M. spretus. We explored whether changing hormone doses would improve response. Females of M. spicilegus were treated with either 2.5 IU of PMSG and 2.5 IU of hCG or with 7.5 IU of PMSG and 7.5 IU of hCG, and the oocytes recovered 14 h post-hCG injection. Three females were used for each of the two treatments and in none of them ovulation occurred; as before, mature follicles were observed in the ovaries. Since M. spicilegus did not respond to the standard superovulation treatment, we examined if oocytes could be collected at later intervals after hCG administration. Females were treated with 5 IU of PMSG followed by 5 IU of hCG 48 h later, and oocytes were collected at different times post-hCG injection. We attempted collection of oocytes from 13 h to 32 h post-hCG and we found that some oocytes could be recovered between 15 h and 22 h with a maximum of oocytes recovered at 19 h (Fig. 2). However, even at this maximum timepoint the number of oocytes collected per female was low (average of 4 oocytes/female), which is not different from the average litter size in this species (3.7 pups/female). With this superovulation result in M. spicilegus, many females would be necessary to obtain
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Fig. 2. Oocytes collected at different times after hCG injection from Mus spicilegus females treated with PMSG and hCG. Females were treated with 5 IU of PMSG followed, 48 h later, by 5 IU of hCG. Oocytes were collected from oviducts at different times after hCG. Number of females used for each treatment is shown in parenthesis. Values are means S.E.M.
enough oocytes to perform an experiment, such as in vitro fertilization. Thus, we searched for alternative procedures to obtain more oocytes. When females were treated with 5 IU of PMSG and, 48 h later, 400 IU of LH, and oocytes recovered 14 h post-LH, we found that ovulation occurred in the three species (Fig. 3) although the number of oocytes recovered from M. spicilegus was low (average: 2 oocytes/female). We examined the effect of different LH doses (250 IU–450 IU) in this species. We found that ovulation occurred with doses between 300 IU and 450 IU of LH (Fig. 4) with a maximum at 350 IU of LH (average: 4.66 oocytes/female). The number of oocytes obtained in M. spicilegus was therefore still low, even with the best LH concentration, and, as in previous treatments, mature (non-ovulated) follicles were present in the ovary.
Fig. 3. Superovulation in three mouse species (Mus musculus, M. spretus and M. spicilegus) after treatment with PMSG and LH. Females of each species were treated with 5 IU of PMSG followed, 48 h later, by 400 IU of LH. Oocytes were collected from oviducts 14 h post-LH administration. Number of females used for each treatment is shown in parenthesis. Values are means S.E.M. Statistically significant differences in the number of oocytes recovered were found between Mus musculus and Mus spicilegus, and between Mus spretus and Mus spicilegus ( p < 0.01).
were not seen among M. musculus or M. spretus oocytes after IVM, whereas an average of 0.7 (3.2%) abnormal oocytes per female were recovered observed in M. spicilegus after IVM. A comparison of the yield of oocytes after superovulation and IVM revealed that the average number of oocytes/female obtained using IVM was significantly higher than that in any of the superovulation treatments; this difference was consistent for each of the three species (Fig. 5). Comparisons between species showed
3.2. In vitro oocyte maturation To examine the yield of oocytes after IVM in the three species, females were treated with 5 IU of PMSG and, 48 h later, COCs were released from the ovaries by follicular puncture and incubated during 17 h for IVM. The average number of COCs/female collected from the three species was 25.7, 19.3 and 11.2 per female in M. musculus, M. spretus and M. spicilegus, respectively. After IVM, 23.3 mature oocytes/female were obtained in M. musculus (maturation rate: 93%), 17.0 mature oocytes/female in M. spretus (maturation rate: 86%), and 10.1 mature oocytes/female in M. spicilegus (maturation rate: 91%) (Table 1). Abnormal oocytes
Fig. 4. Oocytes collected from Mus spicilegus females treated with PMSG and different LH concentrations. Females were treated with 5 IU of PMSG followed, 48 h later, by different concentrations of LH. Oocytes were collected 14 h post-LH administration. Number of females used for each treatment is shown in parenthesis. Values are means S.E.M. There were no significant differences in the number of oocytes obtained with different doses of LH.
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that for both superovulation and IVM there were significant differences in the average numbers of oocytes per female, with M. musculus always yielding the highest number of oocytes/female and M. spicilegus the lowest. 3.3. In vitro fertilization We carried out in vitro fertilization to assess the ability of the superovulated and in vitro matured oocytes to undergo fertilization in the three species. In M. musculus percentages of fertilization were 85%, 84% and 87% for oocytes recovered using PMSG/hCG, PMSG/LH and PMSG/IVM treatments, respectively (Fig. 6A). In M. spretus percentages of fertilization were 83%, 84% and 84% in PMSG/hCG, PMSG/LH and PMSG/IVM treatments, respectively (Fig. 6B). Finally, in M. spicilegus percentages were 85%, 87% and 84% in PMSG/hCG, PMSG/LH and PMSG/IVM treatments, respectively (Fig. 6C). In summary, the percentage of fertilization was similar for the three treatments and between the three species, and there were no statistical differences between any of them. 4. Discussion
Fig. 5. Superovulation and in vitro oocyte maturation in three mouse species (Mus musculus, M. spretus and M. spicilegus). Females of each species were treated with either 5 IU PMSG and 5 IU hCG or with 5 IU PMSG and 400 IU (M. musculus and M. spretus) or 350 IU of LH (M. spicilegus). The interval between hormone administrations was 48 h. Oocytes were collected 14 h after hCG or LH (M. musculus and M. spretus), 19 h after hCG (M. spicilegus) or 14 h after LH (M. spicilegus); these intervals were found to yield the maximum number of oocytes. Immature oocytes were collected by follicular puncture 48 h after 5 IU PMSG injection and cultured for in vitro maturation (see Section 2 for details). (A) Oocyte yield in M. musculus. The number of oocytes/female was significantly higher after in vitro maturation ( p < 0.05). (B) Oocyte yield in M. spretus. The number of oocytes/female was significantly higher after in vitro maturation ( p < 0.03). (C) Oocyte yield in M. spicilegus. The number of oocytes/ female was significantly higher after in vitro maturation ( p < 0.01). Number of females used for each treatment is shown in parenthesis. Values are means S.E.M.
The results of this study on different mouse species (M. musculus, M. spretus and M. spicilegus) reveal differences in superovulatory response between closely related species of mice with one of these species (M. spicilegus) not responding to conventional superovulatory treatments employing PMSG and hCG or LH. Results also showed that oocytes from the three species can be consistently obtained after PMSG stimulation and in vitro maturation. Regardless of the method used to obtain mature oocytes, a high proportion of oocytes underwent fertilization in vitro in the three species and no differences were found either between treatments or between species in fertilization rates. The laboratory mouse is a commonly used animal model in reproductive research. A supraphysiological number of oocytes can be readily obtained using a variety of methods [46,47] and such oocytes can be fertilized either in vivo or in vitro. The mouse model is particularly important in studies aimed at elucidating mechanisms involved in sperm–oocyte recognition, which are critical to our understanding of reproductive isolation among species. To fully benefit from the possibility of incorporating other related mouse species in order to undertake comparative studies, we have characterized superovulatory responses in three mouse species and examined the yield of oocytes via in vitro maturation.
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Table 1 In vitro oocyte maturation in three mouse species (Mus musculus, M. spretus and M. spicilegus) Species Mus musculus Mus spretus Mus spicilegus
Number of COCs/female a
25.7 1.2 19.3 1.2a 11.2 0.7c
Number of mature oocytes/female a
23.3 0.9 17.0 1.1 b 10.1 0.9 c
% Maturation 92.6 1.4a 86.2 2.1a 90.8 1.8a
Females of each species were treated with 5 IU of PMSG and, after 48 h, cumulus–oocyte complexes (COCs) were recovered by follicular puncture and incubated under in vitro maturation conditions. Oocytes were assessed after 17 h of in vitro culture. See Section 2 for details. Number of females used for experiments were 3, 3 and 6 for M. musculus, M. spretus and M. spicilegus, respectively. Values are means S.E.M. Different letters within columns indicate statistical differences ( p < 0.05).
Fig. 6. In vitro fertilization of oocytes collected after superovulation or in vitro maturation in three mouse species (Mus musculus, M. spretus and M. spicilegus). Oocytes were obtained after treatment with PMSG/hCG, PMSG/LH or PMSG followed by in vitro maturation. (A) Mus musculus; (B) Mus spretus; C) Mus spicilegus. Number of females used for each treatment is shown in parenthesis. Values are means S.E.M. There were no significant differences within or among species in fertilization rates of oocytes obtained using different treatments.
When mature females from three mouse species (M. musculus, M. spretus and M. spicilegus) were treated with a combination of 5 IU of PMSG and 5 IU of hCG, differences in number of oocytes collected per female were found between species. Differences were also found between species when 5 IU of PMSG and 400 IU of LH were employed. Superovulation took place in both M. musculus and M. spretus but no or very limited response was seen in M. spicilegus. Examination of the ovaries of M. spicilegus females showed that response to PMSG did take place because mature follicles were present, indicating that superovulation was not occurring due to a lack of response to the dose of hCG or LH or the timing of oocyte collection employed. This phenomenon resembles the situation in the Chinese hamster where females respond poorly to hCG, contrary to the situation in the Syrian (golden) hamster [19]. We were unable to substantially increase the ovulatory response in M. spicilegus by modifying either hCG or LH doses. When the interval of oocyte collection after hGC injection was extended some oocytes could be recovered in this species but the response was not above the natural ovulatory rate and unruptured follicles still remained in the ovary. Due to the poor results obtained after extending the interval for oocyte collection after hCG injection, we did not attempt a similar approach with LH. Several factors may account for differences in response of the three species and, in particular, for the poor response of M. spicilegus females. One possibility is that the different species respond in different ways to the hormonal treatments. Variations in ovarian response to exogenous gonadotrophins may be due to genetic differences and this has been reported for various strains of laboratory mice [22,23,27,28,48,49] There are differences between the three mouse species in their average litter sizes, which most likely is a reflection of differences in average ovulation rate. There was an agreement between the average litter size in the three species and the superovulatory response observed after hormonal treatments, with M. musculus having the
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larger litter size and the highest response to exogenous hormones, with M. spicilegus representing the lowest end of the range. However, the extremely limited response of M. spicilegus suggests that genetic factors may not entirely account for differences between these species. Another possible explanation to the low response following hormone treatment in M. spicilegus could be related to stress factors, as it has been shown that stressors can affect ovulation in mammals through their action on the hypothalamus–pituitary–gonadal axis [50–52]. M. spicilegus might be more sensitive to stress conditions than the other species used in this study. Finally, an intriguing possibility is that sperm competition levels may influence the sensitivity of females, so that in species in which females are highly promiscuous (such as M. spicilegus) ovulation does not respond to a number of stimuli which are effective in other species. It is not clear how these limitations in superovulation could be overcome in M. spicilegus but future work should consider new approaches such as the use of GnRH agonist [8,9] or inhibin antiserum [10] to enhance numbers of ovulated oocytes. An alternative solution may lie in the use of females whose oestrous cycles are monitored in advance and PMSG is injected on a given day of the cycle [34,35], instead of injections on any day of the oestrous cycle, as performed in this study. In any case, this may provide a limited option since repeated handling of females may generate considerable stress and this may outbalance the benefits of a programmed PMSG administration. Since treatment with PMSG consistently resulted in high numbers of mature follicles (despite lack of ovulation in one species), we explored the possibility of increasing the yield of oocytes by means of IVM. In all three species, the number of mature oocytes obtained using IVM was higher than that obtained using superovulation. The yield of oocytes increased by 30% in M. musculus, 38% in M. spretus and 120% in M. spicilegus. As found in other species, IVM is thus a tool to increase the reproductive potential of a female allowing the recovery of follicles whose destiny is the atresia and facilitates the rescue of gametes from females with difficulties to reproduce. However, cytoplasmic and molecular parameters may differ between in vitro matured and in vivo (i.e., ovulated) matured oocytes [53–56]. This was clearly not the case in the present study because nuclear maturation and fertilization rates were very similar between superovulated (in vivo matured) and in vitro matured oocytes, suggesting that the protocols used were able to support in vitro maturation and fertilization to similar extents. Most of the oocytes included in culture progressed to
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metaphase II and were fertilized after co-incubation with homologous spermatozoa, suggesting that cytoplasmic maturation was able to support pronucleus formation and did not differ between the various treatments tested (PMSG/hCG, PMSG/LH, PMSG plus IVM). Further research will be needed to evaluate the ability of these oocytes to cleave and progress to the blastocyst stage. Although fertilization rates were high in all cases, it has to be borne in mind that in vitro matured oocytes were not exposed to oviductal fluids and that this may lead to subtle differences in the properties of the oocyte when interacting with spermatozoa. The zona pellucida from ovulated oocytes contains a ligand recognized by spermatozoa which is absent in the ovarian zona [57]. This sperm-binding ligand in the zona pellucida of ovulated oocytes is likely derived from oviductal secretions [58]. Several studies have examined the potential role of oviductal glycoproteins that bind to the zona pellucida at ovulation [59,60]. The possibility of obtaining oocytes from various mouse species by either superovulation or in vitro maturation, i.e., with or without exposure to oviductal fluids, may allow for a comparative analysis of factors important for sperm– oocyte interaction. In conclusion, oocytes from these three species of mice can be consistently obtained via PMSG stimulation and in vitro maturation and, for two of these species, also by using PMSG/hCG or PMSG/LH stimulation. A high proportion of oocytes thus obtained is capable of undergoing fertilization. These results would allow future studies on gamete interaction in these closely related species and to address the role of sexual selection in gamete compatibility. Acknowledgements Funding was provided by the Spanish Ministry of Education and Science. JM-C was supported by a studentship from the Ministry of Science and Education. RG and CC were supported by studentships from the I3P-CSIC Programme. ERSR is the recipient of a Royal Society Wolfson Research Merit Award. References [1] Nagy A, Gertsenstein M, Vintersten K, Behringer T. Manipulating the mouse embryo: a laboratory manual, 3rd ed., Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2002. [2] Bonhomme F, Gue´net JL. The wild house mouse and its relatives. In: Lyon MF, Searle AG, editors. Genetic variants and strains of the laboratory mouse. Oxford: Oxford University Press; 1989. p. 649–62.
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[21] Salvetti P, Theau-Cle´ment M, Beckers JF, Hurtaud J, Gue´rin P, Neto V, et al. Effect of the luteinizing hormone on embryo production in superovulated rabbit does. Theriogenology 2007;67:1185–93. [22] Vergara GJ, Irwin MH, Moffatt RJ, Pinkert CA. In vitro fertilization in mice: strain differences in response to superovulation protocols and effect of cumulus cell removal. Theriogenology 1997;47:1245–52. [23] Byers SL, Payson SJ, Taft RA. Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs). Theriogenology 2006;65:1716–26. [24] Corbin TJ, McCabe JG. Strain variation of immature female rats in response to various superovulatory hormone preparations and routes of administration. Contemp Top Lab Anim Sci 2002;41: 18–23. [25] Popova E, Bader M, Krivokharchenko A. Strain differences in superovulatory response, embryo development and efficiency of transgenic rat production. Transgenic Res 2005;14: 729–38. [26] Murr SM, Geschwind II, Bradford GE. Plasma LH and FSH during different oestrous cycle conditions in mice. J Reprod Fertil 1973;32:221–30. [27] Durrant BS, Eisen EJ, Ulberg LC. Ovulation rate, embryo survival and ovarian sensitivity to gonadotrophins in mice selected for litter size and body weight. J Reprod Fertil 1980;59:329–39. [28] Schmidt PM, Monfort SL, Wildt DE. Pregnant mares serum gonadotropin source influences fertilization and fresh or thawed embryo development, but the effect is genotype specific. Gamete Res 1989;23:11–20. [29] Kagabu S. The influence of age on the number of non-atretic follicles classified based on follicular size in the rat ovary. Exp Anim 1986;35:165–7. [30] Ozgunen KT, Erdogan S, Mazmanoglu N, Pamuk I, Lologlu G, Ozgunen T. Effect of gonadotrophin dose on oocyte retrieval in superovulated BALB/C mice. Theriogenology 2001;56:435–45. [31] Kagabu S, Umezu M. Variation with age in the numbers of ovulated ova and follicles of Wistar–Imamichi adult rats superovulated with eCG-hCG. Exp Anim 2006;55:45–8. [32] Lang DR, Lamond DR. Some factors affecting the response of the immature mouse to PMS and hCG. J Endocrinol 1966;34:147–54. [33] Andersen CY, Ziebe S, Guoliang X, Byskov AG. Requirements for human chorionic gonadotrophin and recombinant human luteinizing hormone for follicular development and maturation. J Assist Reprod Genet 1999;16:425–30. [34] Tarı´n JJ, Pe´rez-Albala´, Cano A. Stage of the estrous cycle at the time of pregnant mare’s serum gonadotropin injection affects the quality of ovulated oocytes in the mouse. Mol Reprod Dev 2002;61:398–405. [35] Tarı´n JJ, Pe´rez-Albala´ S, Go´mez-Piquer V, Hermenegildo C, Cano A. Stage of the estrous cycle at the time of pregnant mare’s serum gonadotropin injection affects pre-implantation embryo development in vitro in the mouse. Mol Reprod Dev 2002;62: 12–39. [36] Heikinheimo O, Gibbons WE. The molecular mechanisms of oocyte maturation and early embryonic development are unveiling new insights into reproductive medicine. Mol Hum Reprod 1998;4:745–56. [37] Zhang X, Rutledge J, Armstrong DT. Studies on zona hardening in rat oocytes that are matured in vitro in a serum-free medium. Mol Reprod Dev 1991;28:292–6.
J. Martı´n-Coello et al. / Theriogenology 70 (2008) 1004–1013 [38] Anderiesz C, Trounson AO. Fertilization and early embryology: the effect of testosterone on the maturation and developmental capacity of murine oocytes in vitro. Hum Reprod 1995;10: 2377–81. [39] Miki H, Ogonuki N, Inoue K, Baba T, Ogura A. Improvement of cumulus-free oocyte maturation in vitro and is application to microinsemination with primary spermatocytes in mice. J Reprod Dev 2006;52:239–48. [40] Gomendio M, Martin-Coello J, Crespo C, Magan˜a C, Roldan ERS. Sperm competition enhances functional capacity of mammalian spermatozoa. PNAS 2006;103:15113–7. [41] Van der Auwera I, Pijnenborg R, Koninckx PR. The influence of in vitro culture versus stimulated and untreated oviductal environment on mouse embryo development and implantation. Hum Reprod 1999;14:2570–4. [42] Cecconi S, Rossi G, Palmerini MG. Mouse oocyte differentiation during antral follicle development. Microsc Res Tech 2006;69: 408–14. [43] Garza F, Shaban MA, Terranova PF. Luteinizing hormone increases the number of ova shed in the cyclic hamster and guinea-pig. J Endocrinol 1984;101:289–98. [44] Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985;44:493–8. [45] Kawamura K, Kawamura N, Mulders SM, Gelpke DS, Hsueh AJW. Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos. PNAS 2005;102:9206–11. [46] Legge M, Sellens MH. Optimization of superovulation in the reproductively mature mouse. J Assist Reprod Genet 1994;11: 312–8. [47] Mun˜oz I, Rodriguez de Sadia C, Gutierrez A, Blanquez MJ, Pintado B. Comparison of superovulatory response of mature outbred mice treated with FSH or PMSG and development potencial of embryos produced. Theriogenology 1994;41: 907–14. [48] Lubritz DL, Eisen EJ, Robison OW. Effect of selection for litter size and body weight on hormone-induced ovulation rate in mice. J Anim Sci 1991;69:4299–305.
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[49] Spearow JL, Barkley M. Genetic control of hormone-induced ovulation rate in mice. Biol Reprod 1999;61:851–6. [50] Brann DW, Mahesh VB. Role of corticosteroids in female reproduction. FASEB J 1991;5:2691–8. [51] Tilbrook AJ, Turner AI, Clarke IJ. Effects of stress on reproduction in non-rodent mammals: the role of glucocorticoids and sex differences. Rev Reprod 2000;5:105–13. [52] Breen KM, Karsch FJ. New insights regarding glucocorticoids, stress and gonadotropin suppression. Front Neuroendocrinol 2006;27:233–45. [53] Eppig JJ, O’Brien MJ. Comparison of preimplantation developmental competence after mouse oocyte growth and development in vitro and in vivo. Theriogenology 1998;49:415–22. [54] Kim DH, Ko DS, Lee HC, Lee HJ, Park WI, Kim SS, et al. Comparison of maturation, fertilization, development, and gene expression of mouse oocytes grown in vitro and in vivo. J Assist Reprod Genet 2004;21:233–40. [55] Liu XY, Mal SF, Miao DQ, Liu DJ, Bao S, Tan JH. Cortical granules behave differently in mouse oocytes matured under different conditions. Hum Reprod 2005;20(12):3402–13. [56] Moon JH, Jee BC, Ku SY, Suh CS, Kim SH, Choi YM, et al. Spindle positions and their distributions in in vivo and in vitro matured mouse oocytes. Hum Reprod 2005;20:2207–10. [57] Rodeheffer C, Shur BD. Identification of a novel, ZP3-independent egg coat ligand that facilitates initial sperm–egg adhesion. Development 2004;131:503–12. [58] Lyng R, Shur BD. Sperm–egg binding requires a multiplicity of receptor–ligand interactions: new insights into the nature of gamete receptors derived from reproductive tract secretions. In: Roldan ERS, Gomendio M, editors. Spermatology. Nottingham: Nottingham University Press; 2007. p. 335–51. [59] Malette B, Paquette Y, Merlen Y, Bleau G. Oviductins possess chitinase- and mucine-like domains: a lead in the search for the biological function of these oviduct-specific ZP-associating glycoproteins. Mol Reprod Dev 1995;41:384–97. [60] Verhage HG, Mavrogianis PA, O’Day-Bowman MB, Schmidt A, Arias EB, Donnelly KM, et al. Characteristics of an oviductal glycoprotein and its potential role in the fertilization process. Biol Reprod 1998;58:1098–101.
Theriogenology 70 (2008) 1004–1013 www.theriojournal.com
Superovulation and in vitro oocyte maturation in three species of mice (Mus musculus, Mus spretus and Mus spicilegus) J. Martı´n-Coello a, R. Gonza´lez a, C. Crespo a, M. Gomendio a, E.R.S. Roldan a,b,* a
Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC), c/Jose´ Gutie´rrez Abascal 2, 28006 Madrid, Spain b Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 0TU, UK Received 17 April 2008; received in revised form 5 June 2008; accepted 8 June 2008
Abstract Mouse oocytes can be obtained via superovulation or using in vitro maturation although several factors, including genetic background, may affect response. Our previous studies have identified various mouse species as models to understand the role of sexual selection on the evolution of sperm traits and function. In order to do comparative studies of sperm–oocyte interaction, we sought reliable methods for oocyte superovulation and in vitro maturation in mature females of three mouse species (genus Mus). When 5 IU pregnant mare’s serum gonadotrophin (PMSG) and 5 IU human chorionic gonadotrophin (hCG) were injected 48 h apart, and oocytes collected 14 h post-hCG, good responses were obtained in Mus musculus (18 1.3 oocytes/female; mean S.E.M.) and Mus spretus (12 0.8), but no ovulation was seen in Mus spicilegus. Changes in PMSG or hCG doses, or longer post-hCG intervals, did not improve results. Use of PMSG/luteinizing hormone (LH) resulted in good responses in M. musculus (19 1.2) and M. spretus (12 1.1) but not in M. spicilegus (5 0.9) with ovulation not increasing with higher LH doses. Follicular puncture 48 h after PMSG followed by in vitro maturation led to a high oocyte yield in the three species (M. musculus, 23 0.9; M. spretus, 17 1.1; M. spicilegus, 10 0.9) with a consistently high maturation rates. In vitro fertilization of both superovulated and in vitro matured oocytes resulted in a high proportion of fertilization (range: 83–87%) in the three species. Thus, in vitro maturation led to high yields in all three species. These results will allow future studies on gamete interaction in these closely related species and the role of sexual selection in gamete compatibility. # 2008 Elsevier Inc. All rights reserved. Keywords: Superovulation; In vitro maturation; Oocyte; Sperm; Mouse
1. Introduction A reliable supply of large numbers of high quality oocytes, with a good competence for fertilization and development, is an important requisite both for basic reproductive studies and for applied research on assisted
* Corresponding author at: Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC), c/Jose´ Gutie´rrez Abascal 2, 28006 Madrid, Spain. Tel.: +34 91 411 13 28x1245; fax: +34 91 564 50 78. E-mail address: [email protected] (E.R.S. Roldan). 0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.06.002
reproductive techniques. Basic studies require a good oocyte supply to investigate female gamete biology and sperm–egg interactions, providing valuable information about the underlying physiological and cellular mechanisms, as well as about the evolution of species-specificity and reproductive barriers. In the context of assisted reproduction, oocyte supply is crucial for the preservation of genetic resources, conservation of endangered species, transgenesis, or cloning by nuclear transfer, either for reproductive purposes or for the generation of embryonic stem cells to be used in regenerative medicine. Thus, the ability to collect as many unfertilized eggs as possible, and at any time of the day, is an essential tool
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needed for experimental animals, domestic species and endangered wildlife. There are standardized procedures for oocyte superovulation and collection in the laboratory mouse [1], but these may not be applicable to other species. Interest on studies of comparative gamete biology has led to the use of wild mouse species introduced to laboratory conditions [2] and this, in turn, has generated the need to develop new techniques of oocyte collection. Oocyte growth occurs primarily in preantral follicles where the oocyte is arrested in prophase I of meiosis. In a subsequent stage, follicles are selected under the action of follicle stimulating hormone (FSH) and they undergo an increase in size and specialization of granulosa cells into cumulus and mural granulosa cells. The final stage of follicle maturation involves the period between luteinizing hormone (LH) surge and ovulation, and during this time, the oocytes resume meiosis (which arrests at metaphase II) and complete their cytoplasmic maturation; parallel processes in the follicle involve cumulus oophorus expansion, follicle luteinization, and breakdown of the follicle basal lamina [3–5]. These processes can be stimulated with exogenous gonadotrophins and can be supported, in part, during in vitro culture. In mice, about 8–12 ova are released at ovulation, depending on the strain. These numbers can be substantially increased by using exogenous hormones, thereby reducing the animals needed for experimentation [1]. Two main approaches can be used to obtain mature oocytes, namely superovulation (i.e., maturation in vivo) and maturation in vitro. Superovulation involves hormonal treatments to induce follicular development and oocyte release [1,6,7]. Hormones used to induce follicular development are FSH, pregnant mare’s serum gonadotrophin (PMSG) or human menopausal gonadotrophin (HMG). Hormones used to induce follicular rupture and oocyte release include LH and human chorionic gonadotrophin (hCG). Alternatively, hormones such as fertirelin acetate, an LH-releasing hormone analogue, have been used to successfully induce superovulation in mice [8,9]. Induction of superovulation in this species has also relied on the administration of inhibin antiserum [10]. Superovulation with a PMSG/hCG combination is known to induce ovulation in the mouse at any stage to the oestrous cycle [11,12]. This hormonal treatment has also been used to induce superovulation in various rodent species including rats [13,14], Syrian (golden) hamster [15], Siberian (Djungarian) hamster [16] and vesper mice [17,18], but it may sometimes be inefficient in some species such as the Chinese hamster [19]. On
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the other hand, a PMSG/LH combination has been used to reliably induce superovulation in several species [19– 21]. Although superovulation is generally successful for rodent species, it has been found that different strains do not respond in the same way to hormonal treatment (mice: [22,23]; rat: [24,25]). Since superovulation treatments do not increase levels of endogenous gonadotrophins [26], variation in ovarian response to exogenous hormones is likely due to genetic differences between strains [27,28]. Therefore, in order to obtain similar high responses among strains (or species), it may be necessary to adjust hormone concentrations to account for these differences in ovarian response. The response to superovulation treatments varies with age and weight, nutritional status, health and housing conditions of females [1,6]. There are variations with age in the number of ovulated ova per female, with younger females ovulating more oocytes than older ones [6,29–31]. Similarly, females with growth delay, as evidence by lower body weight, tend to yield reduced numbers of oocytes [32]. The dose of administered gonadotrophins [30], the potency [33] and the timing of administration can affect the sensibility to the superovulation treatment and may also be of critical importance for embryo production and development of embryos to the blastocyst stage. Superovulatory response (and subsequent embryo development) can also vary depending on the day of the oestrus cycle when PMSG is administered [34,35]. An alternative approach used to obtain a high number of oocytes involves in vitro maturation (IVM). Studies have established that IVM of oocytes from the germinal vesicle stage to the metaphase II (MII) stage is technically feasible in a range of different mammalian species [20,36], including laboratory animals such as mice and rats [37–39]. The protocol can include an initial administration of hormones such as PMSG or FSH in order to induce development of a higher number of follicles, followed by the maturation of cumulus– oocytes complexes (COCs) obtained from antral follicles in culture medium. In previous work, we have characterized sperm traits and function in various species of mice [40]. In order to test fertilizing capacity of spermatozoa from these wild mice, it is necessary to collect oocytes either via superovulation or in vitro maturation. Preliminary work using standard superovulation protocols for the laboratory mouse revealed considerable differences in response between two species and the inability to obtain a superovulatory response in another one. This highlighted the need to test modifications of this protocol, and to search for alternative means to obtain
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mature oocytes. The objective of this study was to explore conditions for superovulation and in vitro oocyte maturation that would lead to the collection of as many viable mature oocytes as possible in three species of mice from the genus Mus (Mus musculus, Mus spretus and Mus spicilegus) and to evaluate the ability of these oocytes to undergo fertilization in vitro. 2. Materials and methods 2.1. Animals All animal procedures were performed following the Spanish Animal Protection Regulation, RD1201/2005 which conforms to European Union Regulation 2003/ 65. Procedures also adhered to the Guiding Principles for Biomedical Research Involving Animals, as issued by the Council for the International Organizations of Medical Sciences and recommended by the journal. Adult females and males of three species of Murid rodents (M. musculus, M. spicilegus and M. spretus) were purchased from the Laboratoire Ge´nome et Populations, Universite´ de Montpellier II, and kept using standard laboratory mouse conditions, in an environmentally controlled room on a 14 h light:10 h darkness photoperiod under constant temperature and relative humidity conditions. Under these conditions, animals reproduce throughout the year. Animals were provided with food (Harlan Ibe´rica, Barcelona, Spain) and water, both available ad libitum. Animals to be used for experiments were weaned when they were 4 weeks old. Males were kept in individual cages and were used when they were >16 weeks old. After weaning, females were housed together and they were used when they were 12–20 weeks old. We chose to use females of these ages because these wild-derived animals reach maturity at about 3–4 months of age. In addition, we decided against using immature animals for superovulation because one of the aims of future work involves mating or artificial insemination of superovulated animals; immature animals would not be adequate for this type of experimental work. Species used in this study differ in the number of pups per litter (mean S.E.M): M. musculus, 6.9 0.4; M. spretus, 5.7 0.4 and M. spicilegus 3.7 0.5. 2.2. Superovulation Females of three species of mice were subjected to two different hormonal treatments to induce superovulation. In the first hormonal treatment females were injected intraperitoneally with 5 IU of PMSG (Folligon,
Intervet, Madrid, Spain) and 5 IU of hCG (Chorulon, Intervet, Madrid, Spain) 48 h later. Oocytes were recovered 14 h post-hCG. This is the standard superovulation treatment for CD1 and B6D2F1 mice used in our laboratory, as well as by other authors [22,23,41,42]. Due to a limited response in M. spicilegus further studies were carried out to induce superovulation in this species. Females were injected with 2.5 IU of PMSG and 2.5 IU of hCG, or with 7.5 IU of PMSG and 7.5 IU of hCG; in both cases oocyte recovery was performed 14 h posthCG. The effect of time of oocyte recovery after hCG injection was also examined in M. spicilegus females given 5 IU of PMSG and 5 IU of hCG. In the second hormonal treatment, superovulation was induced with PMSG and LH (Sigma, Madrid, Spain) based on results in other species [19,43]. Females of the three species were intraperitoneally injected with 5 IU of PMSG followed 48 h later by 400 IU of LH and oocyte collection 14 h post-LH. In addition, superovulation of M. spicilegus females was attempted with 5 IU of PMSG and different LH concentration (250 IU, 300 IU, 350 IU, 400 IU and 450 IU). After hormonal treatments females were sacrificed by cervical dislocation, reproductive tracts were removed and placed in human tubular fluid (HTF) [44]. Oocyte-containing cumulus masses were released from the ampullae with the aid of a pair of needles under a dissecting microscope and used for in vitro fertilization. 2.3. Oocyte collection from antral follicles and in vitro maturation Immature mouse oocytes were collected from females of the three species 48 h after administration of an intraperitoneal injection of 5 IU of PMSG. In vitro maturation of oocytes was performed as described previously [45]. Ovaries were dissected out and placed in Petri dishes containing M2 medium (Sigma) supplemented with 5% (v/v) heat-inactivated foetal bovine serum (FBS, Gibco, Invitrogen, Barcelona, Spain). COCs were released by follicular puncturing with the aid of a pair of 30 G needles attached to disposable syringes under a stereomicroscope. Only immature oocytes surrounded by compact cumulus cells were selected. After several washes in M2, groups of 20–50 COCs were placed in 4-well culture dishes (Nunclon, Nalgene, Nunc International, Roskilde, Denmark) containing the in vitro maturation medium, covered with 250–300 ml of mineral oil and cultured at 37 8C in 5% CO2 in air and maximum humidity for
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17 h. A volume of 500 ml of minimum essential medium-alpha supplemented with Earle’s salts (MEM-alpha, Gibco, Invitrogen) plus 10 mg/ml streptomycin sulfate, 75 mg/ml penicillin G (Sigma) and 5% (v/v) heat-inactivated FBS was used as maturation medium. Meiosis progression until metaphase II (MII) was confirmed by the presence of the polar body after the incubation period. Maturation rate was recorded and expressed over the total number of oocytes cultured. Mature oocytes were used for in vitro fertilization. 2.4. In vitro fertilization Mouse epididymides were removed from males of the three mouse species and placed in separate 1 ml drops of HTF medium under oil, and incubated at 37 8C in 5% CO2/air for 10 min to allow for sperm dispersion. After incubation, epididymal tissue was discarded and the sperm suspensions were incubated at 37 8C under 5% CO2 in air for capacitation as described before [40]. In vitro fertilization was carried out in 500 ml droplets of HTF medium. Oocytes from each species, obtained after superovulation or after in vitro maturation, were placed in HTF medium. Each sample was inseminated with a final concentration of 0.8–1 106 sperm/ml of capacitated spermatozoa. Gametes were co-incubated for 4 h at 37 8C under 5% CO2 in air. After this period, remaining cumulus cells and attached sperm were removed by washing the oocytes in and out with a fine pipette. Oocytes were then washed three times with HTF medium and placed in 150 ml HTF drops. Fertilization was assessed by pronucleus observation under microscope examination. Fertilization rate was expressed over the number of superovulated or in vitro mature oocytes that were co-incubated with spermatozoa. 2.5. Statistical analysis Variables were transformed to fulfill parametric assumptions: percentages were arcsine-transformed whereas all other variables were log-transformed. Differences between treatments were tested using ANOVA and Tukey tests. A value of p < 0.05 was regarded as indicative of statistical significance. 3. Results 3.1. Superovulation When females were treated with 5 IU of PMSG and 5 IU of hCG, and oocytes recovered 14 h post-hCG, an average of 18 oocytes/female were collected from M.
Fig. 1. Superovulation in three mouse species (Mus musculus, M. spretus and M. spicilegus) after treatment with PMSG and hCG. Females of each species were treated with 5 IU of PMSG followed, 48 h later, by 5 IU of hCG. Oocytes were collected from oviducts 14 h after hCG administration. Number of females used for each treatment is shown in parenthesis. Values are means S.E.M. Differences in the number of oocytes recovered from M. musculus and M. spretus were statistically significant ( p < 0.05).
musculus, and 12 oocytes/female from M. spretus (Fig. 1); on the other hand, oocytes were not recovered from M. spicilegus although there were mature follicles in the ovaries. The proportion of abnormal oocytes observed was low in the species that reponded to superovulation, being on average 1.4 (7.7%) abnormal oocytes per female in M. musculus and 1.3 (15.9%) abnormal oocytes per female in M. spretus. We explored whether changing hormone doses would improve response. Females of M. spicilegus were treated with either 2.5 IU of PMSG and 2.5 IU of hCG or with 7.5 IU of PMSG and 7.5 IU of hCG, and the oocytes recovered 14 h post-hCG injection. Three females were used for each of the two treatments and in none of them ovulation occurred; as before, mature follicles were observed in the ovaries. Since M. spicilegus did not respond to the standard superovulation treatment, we examined if oocytes could be collected at later intervals after hCG administration. Females were treated with 5 IU of PMSG followed by 5 IU of hCG 48 h later, and oocytes were collected at different times post-hCG injection. We attempted collection of oocytes from 13 h to 32 h post-hCG and we found that some oocytes could be recovered between 15 h and 22 h with a maximum of oocytes recovered at 19 h (Fig. 2). However, even at this maximum timepoint the number of oocytes collected per female was low (average of 4 oocytes/female), which is not different from the average litter size in this species (3.7 pups/female). With this superovulation result in M. spicilegus, many females would be necessary to obtain
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Fig. 2. Oocytes collected at different times after hCG injection from Mus spicilegus females treated with PMSG and hCG. Females were treated with 5 IU of PMSG followed, 48 h later, by 5 IU of hCG. Oocytes were collected from oviducts at different times after hCG. Number of females used for each treatment is shown in parenthesis. Values are means S.E.M.
enough oocytes to perform an experiment, such as in vitro fertilization. Thus, we searched for alternative procedures to obtain more oocytes. When females were treated with 5 IU of PMSG and, 48 h later, 400 IU of LH, and oocytes recovered 14 h post-LH, we found that ovulation occurred in the three species (Fig. 3) although the number of oocytes recovered from M. spicilegus was low (average: 2 oocytes/female). We examined the effect of different LH doses (250 IU–450 IU) in this species. We found that ovulation occurred with doses between 300 IU and 450 IU of LH (Fig. 4) with a maximum at 350 IU of LH (average: 4.66 oocytes/female). The number of oocytes obtained in M. spicilegus was therefore still low, even with the best LH concentration, and, as in previous treatments, mature (non-ovulated) follicles were present in the ovary.
Fig. 3. Superovulation in three mouse species (Mus musculus, M. spretus and M. spicilegus) after treatment with PMSG and LH. Females of each species were treated with 5 IU of PMSG followed, 48 h later, by 400 IU of LH. Oocytes were collected from oviducts 14 h post-LH administration. Number of females used for each treatment is shown in parenthesis. Values are means S.E.M. Statistically significant differences in the number of oocytes recovered were found between Mus musculus and Mus spicilegus, and between Mus spretus and Mus spicilegus ( p < 0.01).
were not seen among M. musculus or M. spretus oocytes after IVM, whereas an average of 0.7 (3.2%) abnormal oocytes per female were recovered observed in M. spicilegus after IVM. A comparison of the yield of oocytes after superovulation and IVM revealed that the average number of oocytes/female obtained using IVM was significantly higher than that in any of the superovulation treatments; this difference was consistent for each of the three species (Fig. 5). Comparisons between species showed
3.2. In vitro oocyte maturation To examine the yield of oocytes after IVM in the three species, females were treated with 5 IU of PMSG and, 48 h later, COCs were released from the ovaries by follicular puncture and incubated during 17 h for IVM. The average number of COCs/female collected from the three species was 25.7, 19.3 and 11.2 per female in M. musculus, M. spretus and M. spicilegus, respectively. After IVM, 23.3 mature oocytes/female were obtained in M. musculus (maturation rate: 93%), 17.0 mature oocytes/female in M. spretus (maturation rate: 86%), and 10.1 mature oocytes/female in M. spicilegus (maturation rate: 91%) (Table 1). Abnormal oocytes
Fig. 4. Oocytes collected from Mus spicilegus females treated with PMSG and different LH concentrations. Females were treated with 5 IU of PMSG followed, 48 h later, by different concentrations of LH. Oocytes were collected 14 h post-LH administration. Number of females used for each treatment is shown in parenthesis. Values are means S.E.M. There were no significant differences in the number of oocytes obtained with different doses of LH.
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that for both superovulation and IVM there were significant differences in the average numbers of oocytes per female, with M. musculus always yielding the highest number of oocytes/female and M. spicilegus the lowest. 3.3. In vitro fertilization We carried out in vitro fertilization to assess the ability of the superovulated and in vitro matured oocytes to undergo fertilization in the three species. In M. musculus percentages of fertilization were 85%, 84% and 87% for oocytes recovered using PMSG/hCG, PMSG/LH and PMSG/IVM treatments, respectively (Fig. 6A). In M. spretus percentages of fertilization were 83%, 84% and 84% in PMSG/hCG, PMSG/LH and PMSG/IVM treatments, respectively (Fig. 6B). Finally, in M. spicilegus percentages were 85%, 87% and 84% in PMSG/hCG, PMSG/LH and PMSG/IVM treatments, respectively (Fig. 6C). In summary, the percentage of fertilization was similar for the three treatments and between the three species, and there were no statistical differences between any of them. 4. Discussion
Fig. 5. Superovulation and in vitro oocyte maturation in three mouse species (Mus musculus, M. spretus and M. spicilegus). Females of each species were treated with either 5 IU PMSG and 5 IU hCG or with 5 IU PMSG and 400 IU (M. musculus and M. spretus) or 350 IU of LH (M. spicilegus). The interval between hormone administrations was 48 h. Oocytes were collected 14 h after hCG or LH (M. musculus and M. spretus), 19 h after hCG (M. spicilegus) or 14 h after LH (M. spicilegus); these intervals were found to yield the maximum number of oocytes. Immature oocytes were collected by follicular puncture 48 h after 5 IU PMSG injection and cultured for in vitro maturation (see Section 2 for details). (A) Oocyte yield in M. musculus. The number of oocytes/female was significantly higher after in vitro maturation ( p < 0.05). (B) Oocyte yield in M. spretus. The number of oocytes/female was significantly higher after in vitro maturation ( p < 0.03). (C) Oocyte yield in M. spicilegus. The number of oocytes/ female was significantly higher after in vitro maturation ( p < 0.01). Number of females used for each treatment is shown in parenthesis. Values are means S.E.M.
The results of this study on different mouse species (M. musculus, M. spretus and M. spicilegus) reveal differences in superovulatory response between closely related species of mice with one of these species (M. spicilegus) not responding to conventional superovulatory treatments employing PMSG and hCG or LH. Results also showed that oocytes from the three species can be consistently obtained after PMSG stimulation and in vitro maturation. Regardless of the method used to obtain mature oocytes, a high proportion of oocytes underwent fertilization in vitro in the three species and no differences were found either between treatments or between species in fertilization rates. The laboratory mouse is a commonly used animal model in reproductive research. A supraphysiological number of oocytes can be readily obtained using a variety of methods [46,47] and such oocytes can be fertilized either in vivo or in vitro. The mouse model is particularly important in studies aimed at elucidating mechanisms involved in sperm–oocyte recognition, which are critical to our understanding of reproductive isolation among species. To fully benefit from the possibility of incorporating other related mouse species in order to undertake comparative studies, we have characterized superovulatory responses in three mouse species and examined the yield of oocytes via in vitro maturation.
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Table 1 In vitro oocyte maturation in three mouse species (Mus musculus, M. spretus and M. spicilegus) Species Mus musculus Mus spretus Mus spicilegus
Number of COCs/female a
25.7 1.2 19.3 1.2a 11.2 0.7c
Number of mature oocytes/female a
23.3 0.9 17.0 1.1 b 10.1 0.9 c
% Maturation 92.6 1.4a 86.2 2.1a 90.8 1.8a
Females of each species were treated with 5 IU of PMSG and, after 48 h, cumulus–oocyte complexes (COCs) were recovered by follicular puncture and incubated under in vitro maturation conditions. Oocytes were assessed after 17 h of in vitro culture. See Section 2 for details. Number of females used for experiments were 3, 3 and 6 for M. musculus, M. spretus and M. spicilegus, respectively. Values are means S.E.M. Different letters within columns indicate statistical differences ( p < 0.05).
Fig. 6. In vitro fertilization of oocytes collected after superovulation or in vitro maturation in three mouse species (Mus musculus, M. spretus and M. spicilegus). Oocytes were obtained after treatment with PMSG/hCG, PMSG/LH or PMSG followed by in vitro maturation. (A) Mus musculus; (B) Mus spretus; C) Mus spicilegus. Number of females used for each treatment is shown in parenthesis. Values are means S.E.M. There were no significant differences within or among species in fertilization rates of oocytes obtained using different treatments.
When mature females from three mouse species (M. musculus, M. spretus and M. spicilegus) were treated with a combination of 5 IU of PMSG and 5 IU of hCG, differences in number of oocytes collected per female were found between species. Differences were also found between species when 5 IU of PMSG and 400 IU of LH were employed. Superovulation took place in both M. musculus and M. spretus but no or very limited response was seen in M. spicilegus. Examination of the ovaries of M. spicilegus females showed that response to PMSG did take place because mature follicles were present, indicating that superovulation was not occurring due to a lack of response to the dose of hCG or LH or the timing of oocyte collection employed. This phenomenon resembles the situation in the Chinese hamster where females respond poorly to hCG, contrary to the situation in the Syrian (golden) hamster [19]. We were unable to substantially increase the ovulatory response in M. spicilegus by modifying either hCG or LH doses. When the interval of oocyte collection after hGC injection was extended some oocytes could be recovered in this species but the response was not above the natural ovulatory rate and unruptured follicles still remained in the ovary. Due to the poor results obtained after extending the interval for oocyte collection after hCG injection, we did not attempt a similar approach with LH. Several factors may account for differences in response of the three species and, in particular, for the poor response of M. spicilegus females. One possibility is that the different species respond in different ways to the hormonal treatments. Variations in ovarian response to exogenous gonadotrophins may be due to genetic differences and this has been reported for various strains of laboratory mice [22,23,27,28,48,49] There are differences between the three mouse species in their average litter sizes, which most likely is a reflection of differences in average ovulation rate. There was an agreement between the average litter size in the three species and the superovulatory response observed after hormonal treatments, with M. musculus having the
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larger litter size and the highest response to exogenous hormones, with M. spicilegus representing the lowest end of the range. However, the extremely limited response of M. spicilegus suggests that genetic factors may not entirely account for differences between these species. Another possible explanation to the low response following hormone treatment in M. spicilegus could be related to stress factors, as it has been shown that stressors can affect ovulation in mammals through their action on the hypothalamus–pituitary–gonadal axis [50–52]. M. spicilegus might be more sensitive to stress conditions than the other species used in this study. Finally, an intriguing possibility is that sperm competition levels may influence the sensitivity of females, so that in species in which females are highly promiscuous (such as M. spicilegus) ovulation does not respond to a number of stimuli which are effective in other species. It is not clear how these limitations in superovulation could be overcome in M. spicilegus but future work should consider new approaches such as the use of GnRH agonist [8,9] or inhibin antiserum [10] to enhance numbers of ovulated oocytes. An alternative solution may lie in the use of females whose oestrous cycles are monitored in advance and PMSG is injected on a given day of the cycle [34,35], instead of injections on any day of the oestrous cycle, as performed in this study. In any case, this may provide a limited option since repeated handling of females may generate considerable stress and this may outbalance the benefits of a programmed PMSG administration. Since treatment with PMSG consistently resulted in high numbers of mature follicles (despite lack of ovulation in one species), we explored the possibility of increasing the yield of oocytes by means of IVM. In all three species, the number of mature oocytes obtained using IVM was higher than that obtained using superovulation. The yield of oocytes increased by 30% in M. musculus, 38% in M. spretus and 120% in M. spicilegus. As found in other species, IVM is thus a tool to increase the reproductive potential of a female allowing the recovery of follicles whose destiny is the atresia and facilitates the rescue of gametes from females with difficulties to reproduce. However, cytoplasmic and molecular parameters may differ between in vitro matured and in vivo (i.e., ovulated) matured oocytes [53–56]. This was clearly not the case in the present study because nuclear maturation and fertilization rates were very similar between superovulated (in vivo matured) and in vitro matured oocytes, suggesting that the protocols used were able to support in vitro maturation and fertilization to similar extents. Most of the oocytes included in culture progressed to
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metaphase II and were fertilized after co-incubation with homologous spermatozoa, suggesting that cytoplasmic maturation was able to support pronucleus formation and did not differ between the various treatments tested (PMSG/hCG, PMSG/LH, PMSG plus IVM). Further research will be needed to evaluate the ability of these oocytes to cleave and progress to the blastocyst stage. Although fertilization rates were high in all cases, it has to be borne in mind that in vitro matured oocytes were not exposed to oviductal fluids and that this may lead to subtle differences in the properties of the oocyte when interacting with spermatozoa. The zona pellucida from ovulated oocytes contains a ligand recognized by spermatozoa which is absent in the ovarian zona [57]. This sperm-binding ligand in the zona pellucida of ovulated oocytes is likely derived from oviductal secretions [58]. Several studies have examined the potential role of oviductal glycoproteins that bind to the zona pellucida at ovulation [59,60]. The possibility of obtaining oocytes from various mouse species by either superovulation or in vitro maturation, i.e., with or without exposure to oviductal fluids, may allow for a comparative analysis of factors important for sperm– oocyte interaction. In conclusion, oocytes from these three species of mice can be consistently obtained via PMSG stimulation and in vitro maturation and, for two of these species, also by using PMSG/hCG or PMSG/LH stimulation. A high proportion of oocytes thus obtained is capable of undergoing fertilization. These results would allow future studies on gamete interaction in these closely related species and to address the role of sexual selection in gamete compatibility. Acknowledgements Funding was provided by the Spanish Ministry of Education and Science. JM-C was supported by a studentship from the Ministry of Science and Education. RG and CC were supported by studentships from the I3P-CSIC Programme. ERSR is the recipient of a Royal Society Wolfson Research Merit Award. References [1] Nagy A, Gertsenstein M, Vintersten K, Behringer T. Manipulating the mouse embryo: a laboratory manual, 3rd ed., Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2002. [2] Bonhomme F, Gue´net JL. The wild house mouse and its relatives. In: Lyon MF, Searle AG, editors. Genetic variants and strains of the laboratory mouse. Oxford: Oxford University Press; 1989. p. 649–62.
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