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a Department of Animal Science, University of California Davis, Davis, California 95616
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female high-growth (HG) mice have exhibited poor reproductive capabilities from their inception. While the hg mutation was in the original growth-selected background, the pregnancy rate of the line was approximately 40% lower than in the unselected control line [2]. Once the hg mutation was introgressed into the standard C57BL/6J inbred strain, the fertility was altered, with the HG females experiencing increased ovulation rates and decreased postimplantation survival [13]. Male mice with the hg mutation in the C57BL/6J background experienced lowered fertility with decreased absolute testicular size, sperm production per mouse, and percentage motile spermatozoa [14]. More recent studies revealed that the HG mice (currently a congenic line with the hg mutation in C57BL/6J background) have decreased fertility with an increased incidence of replugging (defined as detection of two consecutive mating plugs without an intervening pregnancy) and recycling (defined as detection of two consecutive mating plugs within an interval of 1–8 days without an intervening pregnancy), and increased intervals between mating and detection of the first mating plug and between mating and conception [15]. The increased incidence of recycling despite the presence of embryos in the oviduct at Day 1 of pregnancy and in the uterus on Days 2 and 3 of pregnancy suggested that the HG females suffer from luteal insufficiency [15].
Corpora lutea (CL) are responsible for the maintenance of early gestation, but in rodents the CL must be rescued by mating-induced prolactin (PRL) surges [16]. Although LH is involved, FSH and PRL together form the necessary luteotropic complex in mice [17]. During early pregnancy, PRL is released in two large daily surges. Nocturnal and diurnal surges in pregnant mice occur before the end of the dark and light periods, respectively [18].
The overall goal was to determine whether the cause of the luteal failure resides in the ovary or the hypothalamic-pituitary unit. The objectives of the present study were to determine 1) whether exogenous progesterone will maintain pregnancy in HG mice, 2) whether PRL injections will rescue the CL and maintain pregnancy in HG females, and 3) whether the ovary of the HG female functions sufficiently to maintain a pregnancy when grafted in a C57 female. To achieve these objectives, we used hormone-supplementation studies and ovarian transplantations, which have been used effectively to answer similar physiological questions in transgenic mice overexpressing GH [19, 20].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The HG line of mice was discovered and developed as described by Bradford and Famula [2]. The hg mutation has since been introgressed into the C57BL/6J background by 9 backcrosses to create congenic mice C57BL/6J-hghg [9]. In this study, animals from generations 16–22 of the congenic line were used. Animals from the inbred C57BL/6J line, generations 184–190, were used as control comparisons. Individual pups were ear-notched between 13 and 15 days of age (day of birth = Day 0). Litters were weaned between 20 and 22 days of age and housed in all-male or all-female groups (4–5 animals per cage) with siblings housed together whenever possible. Weanling animals had feed (Purina [St. Louis, MO] Laboratory Chow 5008: 23.5% protein, 6.5% fat) and water available ad libitum. Mice were housed under controlled conditions of temperature (21°C ± 2°C), humidity (min. 50%), and lighting (14L:10D, lights-on at 0600 h), according to the American Association for Accreditation of Laboratory Animal Care (AAALAC) guidelines for animal care.
Animal Matings
Females were harem-mated (2–3 females/male) at 9–11 wk of age to same-line fertile males of at least 9 wk of age. Before mating, females were examined for the presence of a vaginal septum and eliminated if a vaginal septum was observed. Animals being mated were fed a standard laboratory rodent diet (50:50 mix of Purina Laboratory Chow 5008 [23.5% protein, 6.5% fat] and 5015 [17.5% protein, 11% fat]) and tap water ad libitum. Day of mating (Day 0) was determined by the presence of a vaginal copulatory plug.
Experiment 1
To examine the effects of progesterone injections on maintenance of pregnancy in HG mice, HG females with a demonstrated history of infertility were used. Females that had a sequence of 2 vaginal plugs within an interval of 1–8 days (usually within 6–8 days) were considered infertile. These females received once-daily s.c. injections at 0800 h of 1 mg progesterone suspended in sesame oil (Steris Laboratories, Phoenix, AZ; n = 10) or sesame oil alone (Sigma; n = 10), or received no injections (control; n = 10). Injections were begun on Day 1 postcoitum (Day 0 = day of second plug detection) and continued until the mice were killed on Day 17. Females were anesthetized with sodium pentobarbital (Veterinary Laboratories, Lenexa, KS); the ovaries were examined for presence and number of CL and/or corpora albicantia (CA) while fully vascularized; the females were killed by cervical dislocation; and the entire reproductive tract was removed and the numbers of live, dead, and mummified fetuses were determined.
Experiment 2
The second experiment examined the effects of PRL injections on maintenance of pregnancy in HG mice. Again, only HG females with a demonstrated history of infertility were used for this experiment. After a second mating plug (within 1–8 days of the first plug) was detected, 3 groups of 10 females each received injections of alkaline saline (SAL; pH = 9) or of 100 µg of ovine PRL (NIDDK-oPRL-20 AFP-10677C; FSH content of 0.006% by weight) in alkaline saline (PRL), or were not injected (control; NON). Injections were begun on the morning of Day 1 and were given s.c., twice daily, at 0800 h and 1600 h, to mimic the PRL surges of pregnancy. Injections continued until the evening of Day 5, and the females were killed in the morning of Day 6. Females were anesthetized with sodium pentobarbital; the ovaries, while fully vascularized, were examined for presence and number of CL and/or CA; the females were killed by cervical dislocation; and the entire reproductive tract was removed and the numbers of implantation sites recorded.
Experiment 3
To determine the capacity of the HG ovary to support pregnancy or pseudopregnancy when exposed to the hormonal milieu of pregnancy in a control female, bilateral ovarian transplants were performed between C57 and C57 females, between C57 and HG females, and between HG and C57 females, using 55 recipient animals per group. Females served as both donors and recipients whenever possible. The ovaries were exposed by paralumbar incision under sodium pentobarbital anesthesia and removed by incising the ovarian bursa opposite the ovarian hilum, gently removing the ovary from the ovarian bursa, and excising the ovary by clamping at the ovarian hilum to prevent bleeding. After excision from the donor, the ovaries were held in cold saline until placed inside the ovarian bursa of the ovariectomized recipient. A single stitch of 6–0 polyester (Ethicon, Somerville, NJ) was placed through one side of the incised bursa, through the ovary, and through the opposite side of the incision in the bursa. After 1 wk of recovery while housed individually, females were harem-mated (2–3 females/male) to same-line males and checked daily for vaginal plugs. Vaginal smears were taken daily beginning the day after mating. One estrous cycle was defined as the period from the day nucleated epithelial cells first appeared (i.e., proestrus) to the day preceding the next appearance of nucleated epithelial cells in the vaginal smear, provided that there was a period of leukocytic presence (i.e., diestrus) in between. Estrus was determined by the presence of large, squamous epithelial cells, with or without nuclei. In animals that failed to become pregnant after mating, pseudopregnancy was defined as the presence of leukocytes in the vaginal smear for 9–11 days following the presence of a mating plug. Females were killed on the basis of four classifications depending on their mating activities as follows: 1) females were killed 17–18 days after detection of a mating plug; 2) females were killed 8–9 days after detection of a second mating plug; 3) females were killed 30–31 days after first exposure to a male without the detection of a mating plug; and 4) females were killed 30–31 days after first exposure to a male when numerous plugs (less than 8 days apart) were detected but a pregnancy was not maintained. Females were first anesthetized with sodium pentobarbital, and while fully vascularized the ovaries were examined for maintenance of the ovarian graft, presence of scar tissue, and presence and number of CL and/or CA. The females were then killed by cervical dislocation, the entire reproductive tract was removed, and numbers of live, dead, and mummified fetuses were counted.
Statistics
For comparisons of pregnancy rates, the data were analyzed using independent chi-square tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In experiment 2, daily PRL injections also increased the pregnancy rate (Table 1). Seven of the 10 HG females in the PRL group were pregnant on Day 6 compared with 3 of 10 HG females in the SAL group. In the NON group, 6 of 10 HG females were pregnant. The 70% pregnancy rate in the PRL group was higher than in the SAL group (p < 0.025) but not different from that in the NON group (p > 0.05). Likewise, the SAL females were not different from the NON group (p > 0.05). When data from the SAL and NON groups were combined, the combined group was not different from the PRL group (p > 0.05). Morphologically normal CL were present on ovaries of all pregnant PRL HG females.
In experiment 3, examination of the ovaries of ovarian transplant recipient females at the time of killing revealed that the transplanted ovaries were vascularized, indicating that the ovarian grafts were not rejected by any recipient female in any group. Morphologically normal CL were present on the ovaries of all pregnant graft hosts. Apparently normal and viable fetuses were observed in at least one uterine horn of all pregnant females in all groups.
A summary of the ovarian transplant results is presented in Table 2. Of the 55 recipient animals in each group, 3 animals were discarded from the group in which C57 ovaries were transplanted to C57 recipients because of insufficient healing of surgical sites, and 1 animal was removed from both the HG ovary to C57 recipient and C57 ovary to HG recipient groups because of wounds inflicted on the female by the male. All recipient females in all groups exhibited normal estrous cycles after mating. In the C57 ovary to C57 recipient group, 51 of the 52 females became pregnant after mating. Of the 54 females in the HG ovary to C57 recipient group, 49 females became pregnant after mating. In contrast, only 4 of the 54 females in the C57 ovary to HG recipient group became pregnant after mating. The pregnancy rate of the C57 ovary to HG recipient group was lower than the pregnancy rate of either the C57 ovary to C57 recipient group or the HG ovary to C57 recipient group (p < 0.001), while pregnancy rates in the latter two groups did not differ.
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Of the recipient females that were killed 8–9 days after detection of a second mating plug, 8 of 8 in the C57 ovary to C57 recipient and 6 of 6 in the HG ovary to C57 recipient groups exhibited the second plug 9 or more days after the first plug (Table 3). In the C57 ovary to HG recipient group, 18 of 28 females exhibited a plug for a second time within 1–8 days of the first mating plug and 9 of 28 females exhibited a plug for a second time 9 or more days after the first plug (1 of the 28 females showed a replug more than once, 1 within the 1–8 day category, and 1 within the > 9-day category). More females in the C57 ovary to HG recipient group exhibited the second mating plug within 1–8 days of the first mating plug than either the C57 ovary to C57 recipient or the HG ovary to C57 recipient group (p < 0.001).
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Of the recipient females with numerous mating plugs that were killed 30–31 days after first exposure to a male, all 14 exhibited numerous replugs within 1–8 days of the previous plug. Five of the 14 exhibited a pseudopregnancy as well as numerous replugs within 1–8 days of the previous plug.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Each of the three recipient groups had females that were killed 8–9 days after replugging. This category included both females that exhibited a pseudopregnancy (> 9 days) between the two consecutive mating plugs and females that exhibited a second mating plug within 1–8 days of the first plug (recycle). When the females that were killed 8–9 days after replug were classified into those that exhibited a pseudopregnancy (the expected reason for failed reproductive performance) and those that exhibited a recycle (indicating a female that had luteal failure), only HG recipient females that received C57 ovaries experienced recycling. These results support other observations (unpublished data) that the HG females have a higher incidence of recycling. Furthermore, it indicates that the incidence of recycling was even higher when the HG ovary was replaced by a C57 ovary in the HG recipient than previously seen in the normal HG female, suggesting that the HG ovary compensates for hypothalamic-pituitary deficiencies exhibited by the HG mice. The fact that transplanted ovaries could have a reduced functional mass, which could lead to reduced estrogen levels, is recognized; however, the C57 ovary-into-C57 recipient control group would have experienced the same level of reduced ovarian function, and it did not exhibit recycling.
Daily progesterone injections increased the ability of HG females with a demonstrated history of infertility to maintain pregnancy, which suggests that the infertility caused by increased incidence of recycling was most likely due to luteal failure. This is consistent with results from numerous studies concerning female infertility in several independently derived lines of transgenic mice overexpressing GH [20–22]. Although these different lines of transgenic mice exhibited a higher degree of female infertility than the HG line of mice, similar mechanisms could be involved. A possible mechanism is via increased circulating concentrations of IGF-I observed in both HG mice and transgenic mice overexpressing GH. Rat granulosa cells display high-affinity, low-capacity type I IGF binding sites [23–25], suggesting that IGF-I has a direct effect at the level of the ovary. While IGF-I may act directly at the level of the ovary, its most important role depends on the ability of IGF-I to act in conjunction with pituitary gonadotropins and amplify their effect [26]. Plasma FSH and progesterone concentrations were reduced on Day 7 of gestation in PEPCK-bGH (bovine GH gene fused with the phosphoenolpyruvate carboxykinase promoter) transgenic females [27], indicating that the increased IGF-I levels exhibited by these mice were unable to overcome the adverse effects of suboptimal gonadotropin levels. Although pituitary gonadotropin levels were not measured in the present study, possibly the elevated IGF-I levels could not sustain the reproductive capacity of the HG mice because of altered pituitary gonadotropin levels.
Twice-daily administration of PRL according to a schedule that presumably mimics the daily PRL surges of pregnancy in the mouse [18] increased pregnancy in the HG females compared with the SAL-injected HG females, suggesting that the luteal insufficiency observed in the HG mice was due to insufficient PRL levels. The number of NON females that maintained a pregnancy was not different from the PRL group, but the high percentage of pregnancies observed in the NON group was higher than anticipated based on data collected from the entire HG breeding colony (n = > 2000 females). In the HG breeding colony, slightly fewer than half of the females that exhibited a plug a second time within 1–8 days of the first plug maintained a pregnancy on the second plug (unpublished data). Therefore, the pregnancy maintenance was similar in the NON and PRL groups because HG females that experience luteal insufficiency in one cycle may not experience luteal insufficiency in the following cycle. This suggests that PRL release was insufficient to maintain pregnancy in each estrous cycle. Such a situation is seen in rats: if the male makes few intromissions into the female, the amount of cervical stimulation is low, and the probability for initiation of PRL surges diminishes [28]. Alternatively, the basal PRL levels could be suppressed in the HG females, such that additional stimulation is required to reach the necessary PRL threshold for the establishment and maintenance of pregnancy. One such condition is seen in old cyclic female rats, in which the PRL secretory system experiences a decreased sensitivity to the mating stimulus [29]. The HG females used in the present study were not old, but they could have experienced decreased sensitivity to the mating stimulus. Future studies are necessary to determine whether one of these explanations is the real cause of the problems observed here.
The injection regimen used in the present study was from an established protocol that has been shown to rescue pregnancies in transgenic mice overexpressing GH [19]. The PRL-injected HG females maintained a greater number of pregnancies than the SAL-injected HG females. We have observed repeated instances of defects in the nest-building, nursing, and mothering behaviors in the HG females (unpublished data). Appropriate maternal behavior, such as nest building and nursing/mothering of pups, involves exposure to pregnancy hormones including PRL [30]. A deficit in maternal behavior may suggest abnormal circulating PRL concentrations in HG mice. The results of the PRL supplementation study and the defects in nesting/mothering behavior taken together indicate that abnormal PRL concentrations could be a cause of the luteal insufficiency observed in the HG mice.
Supplementation of HG females with exogenous progesterone or PRL appeared to facilitate development of normal CL morphology. While progesterone does not maintain the morphology of the viable CL, the HG female may experience a problem reaching the threshold level of either progesterone or PRL, and upon administration of exogenous progesterone or PRL, the female's CL forms and functions properly. Alternatively, the progesterone could be converted to estrogens, which have known luteotropic functions. Takayama and Greenwald [31] demonstrated that large daily doses of estrogens restored luteal and plasma levels of progesterone in hypophysectomized, hysterectomized rats. The ovaries of all pregnant progesterone- and PRL-injected HG females displayed grossly normal CL. This observation is similar to findings by Pomp et al. [20], in which oMT1a-oGH (ovine GH gene fused with the ovine metallothionein promoter) transgenic female mice receiving progesterone injections maintained normal CL morphology. Although the CL in this study were not analyzed for progesterone content or histologically evaluated, they appeared to be highly vascularized and comparable in shape and structure to CL on the ovaries of control animals.
In summary, the cause of the poor reproductive performance exhibited by the HG female mice appears to be at the level of the hypothalamic-pituitary unit, not the ovary. The ability of progesterone to maintain pregnancy, the tendency for PRL to support the maintenance of pregnancy, and the defects in maternal behavior all suggest that PRL may play a role in the reproductive inefficiencies observed in the HG mice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Gary B. Anderson, University of California Davis, Animal Science Department, One Shields Ave., Davis, CA 95616. FAX: 530 752 0175; gbanderson{at}ucdavis.edu
Accepted: February 16, 1999.
Received: November 10, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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