Effect of maternal age and parity on preimplantation embryo development and transport in the golden hamster (Mesocricetus auratus) C A Trejo, M C Navarro, G D Ambriz and A Rosado Departamento de Biologı´a de la Reproduccio´n, Universidad Auto´noma Metropolitana – Iztapalapa, Av. Michoaca´n y Purı´sima Col. Vicentina, Iztapalapa, DF, CP 09340, Me´xico
Summary Preimplantation embryo development was studied in the golden hamster (Mesocricetus auratus). Three groups of regularly cycling female hamsters were used: (I) 30 nulliparous young female (NYF) hamsters; (II) 24 nulliparous adult female (NAF) hamsters and (III) 30 multiparous adult female (MAF) hamsters. Female hamsters were mated with male hamsters of proven fertility. Only 15 min were allowed for mating. The moment of ejaculation was registered. Female hamsters were killed from 60 to 69 h after coitus. Corpora lutea were counted in both ovarian surfaces. Oviducts and uterine horns were flushed separately and embryo number, stage of development and distribution were recorded. Adult female hamsters, nulliparous and multiparous, had significant higher ovulation rates than NYF, but their reproductive efficiency was significantly lower. Preimplantation embryo development and transport were highly synchronous in NYF, but not in adults. Morulae were observed in NYF as early as 62–63 h after coitus. In adult female hamsters, significant numbers of morulae were found until 66–67 h. On the contrary, in NYF four-cell embryos were detected only until 60–61 h, while four-cell embryos were found until 64–65 h in NAF, and until 66–67 h in MAF. Embryo transport from the oviduct to the uterus is practically completed at 62–63 h after coitus in NYF, while it is evidently retarded in adult animals. In NYF all eight-cell embryos reached the uterus by 62 h after coitus. In adult female hamsters, both nulliparous and multiparous, a considerable number of eight-cell embryos fail to migrate into the uterus even at 67 h after coitus. Keywords Hamster; embryo development; embryo transport
Our knowledge of the factors that participate in early mammalian embryo development and transport in vivo is still incomplete (Lonergan et al. 1999). Although there is solid evidence that hormonal state of the female hamster participates in the regulation of ovum transport in mammals (Erb & Wynne-Edwards 1993, Croxatto 2002), the differential velocities of oviduct Correspondence: C A Trejo. Email:
[email protected] Accepted 8 December 2004
transport of fertilized and unfertilized ova in several mammalian species (Ortiz et al. 1986) originated the hypothesis that ovum itself is capable of regulating its own development and transport in the early stages of embryo development. In accord with this hypothesis, there have been some recent reports showing that the embryo regulates its own development and transport by the secretion of some active factors like platelet-activating factor (PAF) (Velasquez
r Laboratory Animals Ltd. Laboratory Animals (2005) 39, 290–297
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et al. 2001), some other eicosanoids (Wijayagunawardane et al. 1999) and nitric oxide (Gouge et al. 1998, Chen et al. 2001, Tranguch et al. 2003). Many of the recent studies on mammalian embryo development have been based on carefully designed in vitro – or at least partly in vitro – experiments that must, however, be correlated with carefully obtained in vivo results (Erb & Wynne-Edwards 1993). We have shown that to study with precision the preimplantation development of mammalian embryos in vivo it is necessary: (a) to accurately pinpoint the moment of fertilization, (b) to register the point in the reproductive tract in which embryos at specific stages are located and (c) to understand the physiological variables that may participate in the process (Austin 1956, Mizoguchi & Dukelow 1981, Navarro et al. 2000). From these standpoints, the study of hamster reproductive physiology offers significant advantages: hamster reproduction is easy and convenient on animal facilities; sexual maturity on the female hamster occurs at 4–6 weeks of age (Magalhaes 1970); the female hamster presents an oestrous cycle of four days that is usually normal and easily predictable by external signs (Magalhaes 1970, Barnett & Bavister 1992); pregnancy lasts 16 days and each female hamster gives birth to 9–12 pups. When compared with nulliparous young (two months old) female hamsters, multiparous adult (six months old) female hamsters show significant asynchrony and retard on early embryo development (from two-cell to morulae stages) (Navarro et al. 2000). In this work we decided to extend our studies to adult (eight months old) nulliparous and multiparous female hamsters, studying early embryonic development and oviduct to uterine embryo transport (from the stage of two-cell to the stage of morulae), trying to carefully establish the moment of fertilization and taking into consideration the age and parity of the female hamster (Soderwall et al. 1960, Blaha 1964).
Materials and methods All animals utilized were outbred on our animal facilities from a Harlan Teklad
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HsdHan:AURA strain (Madison, WI, USA). They were housed in windowless rooms with an artificial light–dark cycle (LD ¼ 14:10 h; lights on at 04:00 h), at 251C, 65–70% humidity and 15 changes of air per hour. Tap water and food pellets (Harlan Teklad) were provided ad libitum. Bedding material (softwood fibres) was changed weekly. After weaning all female hamsters were housed (four per cage) in plastic cages (55 33 20 cm), except when, in the case of adult multiparous, isolation was necessary after deliveries. Oestrous cycling was followed in all female hamsters by vaginal cytology and by careful observation of vaginal secretion. Eighty-four female hamsters chosen for regularity of oestrous cycle (at least three four-day regular consecutive cycles) were divided into three experimental groups: (I) 30 nulliparous young female (NYF) hamsters (2–3 months old) weighing 128715 g; (II) 24 nulliparous adult female (NAF) hamsters (eight months old) weighing 168718 g; and (III) 30 multiparous (utilized during their fourth pregnancy), adult female hamsters (MAF) (eight months old). In all cases, 10–12 h after obtaining a clear cytological image of proestrous, the female hamsters showed abundant vaginal secretion (Magalhaes 1970, Mizoguchi & Dukelow 1981). To obtain pregnant animals, giving enough time for sperm capacitation (Kent 1964), the female hamsters were individually caged (3 h after the clear demonstration of proestrous, about 8 h before exhibiting abundant vaginal secretion, and usually 84 h after the last proestrous discharge) (Orsini 1961, Magalhaes 1970) with a male hamster of proven fertility and trained to mount quickly (Magalhaes 1970). Mating was usually performed during the dark phase of the light cycle, but the exact time depended always on the oestrous cycle of the female hamster. Only 15 min were allowed for mating and the occurrence of the ejaculation pattern was directly observed. Fertilization was considered successful if sperm were in the vaginal smear taken 60 min after the mount (Blaha 1964). Female hamsters were differentially responsive to the presence of the male hamster, while all young female hamsters were copulated and fertilized, Laboratory Animals (2005) 39
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seven adult female hamsters (three multiparous and four nulliparous) were not mounted, or not fertilized (no sperm present in the vaginal smear) in the allowed time. The time of ejaculation was carefully registered and lots of female hamsters were killed by neck dislocation from 60 to 69 h after coitus. The corpora lutea (CL) in both ovarian surfaces were counted under 60 magnification (Figure 1). There was no evidence of laterality on CL number between the two ovaries, nor between the sizes of different CL on the same ovary. The genital tract was dissected clean of fat and the oviducts and the uterine horns were separately flushed with saline phosphate buffer (SPB). The flushings were observed under low magnification (Mizoguchi & Dukelow 1981, Nieder & Caprio 1990) and the number, stage of development and distribution of embryos were recorded (Ortiz et al. 1986, Velasquez et al. 2001). Embryo stage was always established as the pair number of blastomeres present or as the pair number immediately above in the case of unpaired number of cells. In the case of eightcell embryos, no distinction was made between compacted or non-compacted embryos. The data were statistically analysed by variance analysis, w2 test, or comparison of means by Student’s t-test. Differences were considered significant when Po0.05.
the sperm cell and therefore the beginning of embryo development has been established (Bavister et al. 1984) to occur between 8 and 10 h after coitus. To normalize our results and make them easily exploitable, we took as zero time for embryo development the time of coitus which in our case was precisely established by visual observation. Table 1 summarizes the general results of the recovery of embryos from the three
Results Exact time of fertilization can be established only by calculation (Blaha 1964, Sato & Yanagimachi 1972, Bavister et al. 1984). The time of penetration of the hamster oocyte by
Figure 1 Ovary of nalliparous young female hamster. Six corpora lutea can be observed in this surface (head of arrows). Magnification 60
Table 1 Total numbers of observed corpora lutea (CL) and embryos recovered from the reproductive tract of young and adult female hamsters Females
n
No. embryos per group
Total number of embryos
Efficiency (%)
Ovulation rate
Young nulliparous Adult nulliparous Adult multiparous
30 24 30
9.271.2w 9.771.6w 8.270.9z
277 233 245
87.1w 77.6z 61.7y
10.670.85w 12.571.18z 13.272.06z
Reproductive efficiency was calculated taking in consideration the number of CL and the total number of embryos recovered from all female hamsters of
each group
Ovulation rate is indicated as the mean7the standard deviation of the data obtained by counting the CL of both ovaries in each female hamster of the
studied groups. No evidence of laterality in CL number between ovaries was found in any of the studied groups Different superscript symbols indicate significant differences (Po0.05) (variance analysis, w2 test)
Laboratory Animals (2005) 39
Hamster embryo development and transport NULLIPAROUS YOUNG FEMALES (NYF) (n = 30)
A
Embryo types recovered at the specified times (percentage)
4 cells Embryos 6 cells Embryos 8 cells Embryos Morulae 100% 75% 50% 25%
60 - 61
62 - 63 64 - 65 66 - 67 Hours Postcoitus
68 - 69
NULLIPAROUS ADULT FEMALES (NAF) (n = 24)
B
Embryo types recovered at the specified times (percentage)
4 cells Embryos 6 cells Embryos 8 cells Embryos Morulae 100% 75% 50% 25%
60 - 61
62 - 63 64 - 65 66 - 67 Hours Postcoitus
68 - 69
MULTIPAROUS ADULT FEMALES (MAF) (n = 30)
C
4 cells Embryos 6 cells Embryos 8 cells Embryos Morulae Embryo types recovered at the specified times (percentage)
groups of female hamsters studied. It is interesting to mention that in spite of the significant higher ovulation rate of the adult female hamsters, both nulliparous and multiparous, their reproductive efficiency was significantly lower than that of the young female hamsters. Embryo development and transport was significantly different in NYF than in adult, nulliparous or multiparous, female hamsters. NYF showed better synchrony of the process of segmentation than adult female hamsters (Figure 2a). In fact, while in NYF there were generally only two stages of segmentation present (exceptionally three in the early hours of development), in NAF (Figure 2b) there were three stages of embryo development until 66–67 h after coitus, and in MAF there were usually three and even four different stages of development at all the studied times (Figure 2c). This is similar to the report of Nieder and Caprio (1990) in the Siberian hamster. Morulae were observed in young female hamsters as early as 62–63 h after coitus, while in adult female hamsters significant numbers of morulae were found until 66–67 h after coitus. On the contrary, in NYF four-cell embryos were detected only at 60–61 h, while significant numbers of embryos in this stage of embryo development were found until 64–65 h in NAF, and until 66–67 h in MAF. Figure 3 shows the percentage of female hamsters with embryos in the uterus at different times after coitus. Embryo transport from the oviduct to the uterus is practically completed at 62–63 h after coitus in NYF, while it is evidently retarded in adult animals. Embryo transport seems to be significantly more efficient in NAF than in MAF in early development (60–65 h after coitus), but this difference is lost at longer times. Figure 4 shows the important differences found in the total number of embryos (six-cell to morulae) recovered in oviduct and uterine flushing from each group of female hamsters at 62–63 h after coitus. A similar distribution pattern was observed at 64–65 h after coitus (data not shown). The smaller number of embryos in MAF is explained by the persistent presence
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100% 75% 50% 25%
60 - 61
62 - 63 64 - 65 66 - 67 Hours Postcoltus
68 - 69
Figure 2 Stages of embryo development between 60–69 h postcoitus. Proestrous female hamsters (about 8 h before exhibiting abundant vaginal secretion, and usually 84 after the last proestrous discharge) were individually caged with a male hamster of proven fertility and trained to mount quickly. Only 15 min were allowed for mating. Pregnant female hamsters were killed hourly by neck dislocation from 60 to 69 h after coitus. The genital tract was dissected clean of fat and the oviducts and the uterine horns were flushed. Embryo stage was always established as the pair number of blastomeres present or as the pair number immediately above in the case of unpaired numbers of cells. In the case of eight-cell embryos no distinction was made between compacted or non-compacted eight-cell embryos. (a) NYF hamsters (b) NAF hamsters (c) MAF hamsters Laboratory Animals (2005) 39
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C A Trejo et al. 60 Total number of embryos per group Utenus Oviduct
YOUNG NULLIPAROUS ADULT NULLIPAROUS ADULT MULTIPAROUS 100
% of females
80
60
40
40 8 cells embryos 20
20 40 60 80
60-61
62-63 64-65 66-67 Hours postcoitus
68-69
Figure 3 Female hamsters with embryos in uterus. The data indicate the percentage of all female hamsters of each group who showed the presence of embryos in both uterine horns at the specified times. Isolated uterine horns were flushed with saline phosphate buffer. The flushings were observed under low magnification and the number of embryos was recorded
of 4-cell embryos in this group of animals (see Figure 2).
Discussion Our data on the time of embryo development in NYF are significantly shorter than the times indicated by some other groups (Austin 1956, Sato & Yanagimachi 1972, Bavister et al. 1984, Ortiz et al. 1986). These differences may be adequately explained by our accuracy in controlling the time of copulation (the moment of male ejaculation during coitus was established by direct observation) (Navarro et al. 2000) and perhaps for the inclusion of compacted eight-cell embryos and morulae, which Austin (1956) and Sato and Yanagimachi (1972) do not distinguish with precision in their works. In addition, the special care taken to establish with precision the time of ovulation (Austin 1956, Magalhaes 1970) allowed us to insure that the time of egg activation occurred 8–10 h after copulation Laboratory Animals (2005) 39
6 cells embryos Morulae
20
0
Young Nulliparous Adult Nulliparous Adult Multiparous
62-63 HOURS AFTER COITUS
Figure 4 Total number of embryos, six-cell to morulae, per group of female hamster found in uterus or in oviduct at 62–63 h postcoitus. After dissection of the genital tract, oviducts and uterine horns were flushed separately with saline phosphate buffer. The flushings were observed under low magnification and the number, stage of development and distribution of embryos were recorded
(Bavister et al. 1984). From our results we may conclude that to accurately establish the time of embryo development in mammals it is necessary: (a) to carefully establish the reproductive characteristics of the female hamsters and the time of ovulation, and (b) to pinpoint by direct observation the time of copulation. The observed decline in fertility and fecundity that accompany aging express the occurrence of multiple processes, including fertilizability of ova, embryo survival, development and transport (Mizoguchi & Dukelow 1981, Bavister et al. 1984, Barnett & Bavister 1992, Bavister 1995). Our results indicate that embryo transport and development differs significantly between young and adult female hamsters. Embryo development is significantly retarded in adult hamsters. This retardation time is more evident in MAF reaching almost 5 h in comparison with NYF. This phenomenon is manifested not only in the persistence of four-cell embryos (even two-cell embryos can be observed in multiparous female hamsters at 62–63 h after coitus) (Navarro et al. 2000), but also in the absence of significant numbers of morulae which in
Hamster embryo development and transport
multiparous female hamsters occurs until 66–67 h after coitus. These results are similar to those of Blaha (1964) who found embryos of four (or less) cells at 63–68 h after ovulation. They amounted to only 1% in young animals (2.5–6 months old), but reached nearly 18% in adult animals (14–18 months old). It has been shown that pregnancy time may be correlated with age. Soderwall et al. (1960) reported that 1month-old female hamsters have a mean gestation period of 373 h, 4-month-old female hamsters gave birth after 378 h of pregnancy, 8-month-old female hamsters have a pregnancy period of 386 h, and 14month-old female hamsters gave birth after 402 h, i.e. they showed a 24–29 h longer pregnancy period than young female hamsters. It is then possible to propose that a significant part of the reported time differences in pregnancy between young and adult female hamsters can be accounted for by differences in the time of early embryo development. The time elapsed between fertilization and implantation, 2–4 days, is remarkably analogous across a broad range of mammalian species. Since these few days between fertilization and implantation have been reported to be of utmost importance (Erb & Wynne-Edwards 1993), the complete understanding of this short period becomes crucial in many aspects of mammalian reproduction. Our results, in accord with the results of Soderwall et al. (1960) and of Parkening and Chang (1976), indicate that, although the ovulation rate in adult hamsters is significantly higher than in NYF hamsters, the reproductive efficiency is significantly higher in NYF (87.1%) than in adults, both nulliparous and multiparous. Of importance is the highly significant difference found in reproductive efficiency between NAF (77.6%) and MAF (61.7%). It has been shown that embryos that cleave rapid and harmoniously have better probabilities of getting efficiently implanted (Edwards 1998, Kubisch et al. 1998, Lonergan et al. 1999). Therefore, the retard and asynchrony in preimplantation embryo development reported in this work in adult, both nulliparous and multiparous female
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hamsters, may be important in explaining the decline in fertility and fecundity that accompany aging (Parkening & Chang 1976, Mizoguchi & Dukelow 1981, Van Kooij et al. 1996). It is important to mention that the data on ovulatory rate and reproductive efficiency obtained from the number of CL can be accepted in the case of the golden hamster because in this, and related animal species (Wynne-Edwards et al. 1987), no more than one generation of CL is present in the ovaries of cycling female hamsters (Greenwald 1985). This is in contrast with the results obtained in some other rodents, like rats and mice, where two, three, or more generations of CL may be simultaneously present from the immediately preceding ovulatory cycles. The number and distribution of embryos in the reproductive tract of the pregnant hamster was very different in young than in adult female hamsters. In young, nulliparous, Djungarian female hamsters, embryo development and transport was reported to be notably harmonious; no embryo of less than eight-cell is transported into the uterus (Nieder & Caprio 1990). This is similar to the observations reported herein in the young golden hamster, but in contrast with our observations in the adult animals in which considerable numbers of six-cell embryos (occasionally even four-cell embryos in MAF hamsters) were found in the uterus. It is also worth observing that while in NYF all eight-cell embryos reached the uterus at 62 h after coitus, a considerable number of eight-cell embryos fail to migrate into the uterus in adult female hamsters, both nulliparous and multiparous, even at times as advanced as 65 h after coitus. Significant decreases in sexual behaviour (Meisel et al. 1988, Meisel & Sterner 1990), modification of body weight (Meisel et al. 1990, Fritzsche et al. 2000) and elevated plasma progesterone levels, due to the increased number and size of CL (Fritzsche et al. 2000), have been reported in groupkept, socially stressed animals. Although it would be difficult to assess the significance of stress-producing conditions during our experiments, we may say that all animals were similarly manipulated, all were Laboratory Animals (2005) 39
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consistently caged in groups of four animals per cage. No undue changes in body weight were observed in any of the groups during the study time. However, stress-producing conditions might explain the decrease in sexual behaviour (Meisel et al. 1988) and the significant increase in the number of CL found in adult individuals (Fritzsche et al. 2000). Although during some time it was believed that the endocrinological activity of the ovary played the determining role in early embryonic development and transport, it has been recently suggested that embryonic derived regulatory signals may be the most important factors controlling preimplantation embryo development and transport (Gouge et al. 1998, Velasquez et al. 2001, Tranguch et al. 2003). Our results provide evidence that age and parity deeply influence embryo development and transport in the normally cycling female hamster. So, our results may not support the hypothesis that preimplantation embryo development and transport is auto-regulated by the embryo itself. References Austin CR (1956) Ovulation, fertilization and early cleavage in the hamster (Mesocricetus auratus). Journal of Royal Microscopical Society 75, 141 Barnett DK, Bavister B (1992) Hypotaurine requirement for in vitro development of golden one-cell embryos into morulae and blastocyst and production of term offspring for in vitro-fertilized ova. Biology of Reproduction 47, 297–304 Bavister BD (1995) Culture of preimplantation embryos: facts and artifacts. Human Reproduction 1, 91–148 Bavister BD, Lorraine M, Lieberman G (1984) Development of preimplantation embryos of the golden hamster in a defined culture medium. Biology of Reproduction 28, 235–47 Blaha GC (1964) Effect of age of the donor and recipient on the development of transferred golden hamster ova. Anatomical Record 150, 413–16 Chen HW, Jiang WS, Tzeng CR (2001) Nitric oxide as a regulator in preimplantation embryo development and apoptosis. Fertility and Sterility 75, 1163–71 Croxatto HB (2002) Physiology of gamete and embryo transport through the fallopian tube. Reproduction Biomedical Online 4, 160–9 Laboratory Animals (2005) 39
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