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Leptin Requirement for Conception, Implantation, and Gestation in the Mouse

Departments of Physiology (N.M.M., J.F.M., C.A.W., M.J.S.) and Medical Genetics (N.D.C.), St. Georges Hospital Medical School, London, United Kingdom SW17 0RE; and Department of Veterinary Basic Sciences, Royal Veterinary College (R.J.S.), London, United Kingdom NW1 0TU

Address all correspondence and requests for reprints to: Dr. Nasser Malik, Department of Physiology, St. Georges Hospital Medical School, Cranmer Terrace, London, United Kingdom SW17 ORE. E-mail: nmalik{at}sghms.ac.uk

	Abstract

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The ob/ob mouse has a complete absence of circulating leptin, resulting in obesity and infertility. Using the minimum daily dose of leptin required to maintain normal body weight and sexual maturation (5 mg/kg, ip), leptin-treated ob/ob females were mated with either wild-type (+/+) or leptin-treated ob/ob males. The leptin treatment continued throughout pregnancy until weaning or was withdrawn at 0.5, 3.5, 6.5, or 14.5 d post coitum (dpc). Normal pregnancy and parturition with pups of normal weight resulted when ob/ob females were mated with +/+ males and leptin treatment was continued throughout pregnancy (6 of 8 pregnancies), to 14.5 dpc (6 of 8 pregnancies), or to 6.5 dpc (9 of 12 pregnancies). Pregnancy did not result when treatment was stopped at 3.5 dpc (1 of 7 pregnancies) or 0.5 dpc (0 of 6 pregnancies). Similar results were obtained when leptin-treated ob/ob females were mated with leptin-treated ob/ob males. The newborn pups failed to survive after birth in groups treated with leptin up to 14.5 and 6.5 dpc despite reinstating leptin at birth. This appeared to be due to a lack of development of the mammary glands. In conclusion, we have shown that leptin is essential for normal preimplantation and/or implantation processes. It is also essential for normal development of the mammary glands, but is not required for pregnancy and parturition once implantation is established.

	Introduction

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THE OBESE (ob) gene encodes leptin, a peptide hormone that is secreted mainly by adipose tissue into the circulation, where it is transported into the central nervous system. Leptin provides information on the size of fat stores to central ob receptors to regulate energy expenditure and food intake (1, 2). In humans and rodents, plasma leptin levels increase dramatically during pregnancy and then fall to prepregnancy levels around birth (3, 4, 5), suggesting a role for leptin in pregnancy. In addition, the demonstration of leptin and ob gene expression in the placenta and fetus (6) suggests a role for leptin in fetal growth and development.

Much emphasis has been placed on leptin’s role in reproduction (7, 8, 9), even though there is evidence that leptin might not be required throughout pregnancy (10). A natural knockout model of leptin is the ob/ob mouse, which has a point mutation in the ob gene that results in the total absence of circulating leptin. As a result ob/ob mice are obese, diabetic, hypogonadal, and infertile and cannot reproduce unless treated with exogenous leptin. In a previous study (10) the fertility of ob/ob female and male mice was restored by treatment with leptin before they were mated, and leptin treatment was then withdrawn at various times after coitus. Surprisingly, this showed that after coitus, leptin was not required for pregnancy to proceed to term with the birth of a litter of ob/ob neonates. However, relatively high replacement doses of human leptin were used, which may have extended the duration of action well into pregnancy.

The aim of this study, using recombinant murine leptin at the minimum dose necessary to produce fertile ob/ob mice of normal body weight, was to identify the critical periods of leptin requirement for conception, implantation, and gestation in the mouse. Two approaches were used to produce fertile ob/ob mice. In the first instance the genotype of the neonates was determined, and the ob/ob neonates were injected with daily doses of leptin, adjusted to produce the same weight gain and sexual maturation as lean littermates. The second approach involved starting leptin treatment at 7 wk of age, when the phenotype of the ob/ob mice had become apparent. Fertility was restored in female and male ob/ob mice by both methods; however, the second approach required less leptin and fewer injections and avoided the stress of genotyping from tail tips. The second approach was therefore used to determine the critical periods of leptin requirement during pregnancy.

Initially, fertile ob/ob female mice were mated with lean (+/+) males to ensure that all fetuses were capable of producing leptin, and leptin treatment was continued or withdrawn at various times during pregnancy. Subsequently, ob/ob females were mated with fertile ob/ob males to produce ob/ob fetuses so that the only source of leptin in the pregnant female was the injected, exogenous leptin.

	Materials and Methods

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Leptin treatment of ob/ob mice
Mice of the Aston strain bearing the ob/ob (R-105X) mutation were housed at the St. Georges Hospital Medical School animal care facility in a temperature- and humidity-controlled room with a 12-h light, 12-h dark cycle (lights on at 0700 h). The mice were caged individually and weighed daily. Food (Lillico Ltd., Surrey, UK) and water were provided ab libitum to all mice. Recombinant murine leptin (Insight Biotechnology Ltd., Middlesex, UK) was dissolved in 5 mM sodium citrate (pH 4), and the pH was adjusted to 7.1–7.2. This was then diluted to the appropriate concentration in sterile saline. Leptin was administered by ip injection, either once daily (1700 h) or twice daily (0900 and 1700 h). The injection volume did not exceed 0.6 ml. All wild-type +/+ males or leptin-treated ob/ob males were test-mated to wild-type females to prove their fertility before being mated to ob/ob females.

Protocol A
Litters of 10- to 12-d-old neonates produced by heterozygote (ob/+) matings were genotyped for the R-105X mutation. DNA was extracted from 0.4 cm of the tail tip using the DNeasy kit (QIAGEN, Valencia, CA) and genotyped as described previously (11). Male and female ob/ob mice were distinguished from homozygotic lean (+/+) males and females by earmarking them.

Females. At weaning, each ob/ob female was housed with a +/+ female littermate, both were weighed daily, and the ob/ob female was injected with murine leptin. The dose of leptin was gradually increased from 0.5 mg/kg to give the same weight gain as the reference +/+ female. At 8 wk of age the ob/ob female was removed and mated with a lean +/+ male. The female was checked twice daily for the presence of a copulatory plug. The minimum effective dose was defined as the leptin dose producing a rate of weight gain comparable to that of the +/+ littermate, which also resulted in a pregnancy that went to term. This dose was found to be 2 mg/kg twice daily from 21–45 d of age, followed by a dose of 3 mg/kg twice daily. After detection of a copulatory plug, this dose was reduced to a single daily injection of 3 mg/kg.

Males. Weaning ob/ob males were selected and treated as described above. At 8 wk of age each male was mated with a wild-type female. The minimum effective dose required to control weight and produce pregnancies in two wild-type females was the same as the minimum effective dose for females.

Protocol B: females and males
Seven-week-old ob/ob mice produced from double heterozygote matings were selected by phenotype (i.e. body weight of approximately 41 g). They were given 5 mg/kg twice daily for about 8 d until their weight dropped to approximately 35 g. The dose was then reduced to 5 mg/kg once daily. Females were mated with either a +/+ male or an ob/ob male that had been treated similarly with leptin. The female was checked twice daily for the presence of a copulatory plug. Once a plug was found, leptin treatment was continued at 5 mg/kg once daily until weaning or was withdrawn at 0.5, 3.5, 6.5, or 14.5 d post coitum (dpc), counting 0.5 dpc as the day the plug was found. The male was kept with the female until parturition, and the female was checked daily for a plug even after the first plug was observed, as not all first plugs were fertile. When a pregnancy resulted in a litter, leptin treatment of the mother was reinstated during the suckling period. Leptin treatment was stopped if pregnancy did not develop after 40 d of treatment.

Control mice
Lean (+/+) Aston female mice were mated with lean (+/+) Aston males and treated according to protocol B with vehicle (sodium citrate solution, pH 7.1).

Statistics
Fisher’s exact test for a 2-by-5 table was used to test the null hypothesis that there is no relationship between days of leptin exposure during pregnancy and success of pregnancy.

Logistic regression was used to test the null hypothesis that there is no difference in pregnancy rates when an ob/ob female is mated with a +/+ male compared with when it is mated to an ob/ob male provided leptin treatment is given for 6.5 dpc or longer.

	Results

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For an ob/ob female to conceive by protocol A took approximately 75 injections requiring approximately 4.3 mg leptin. To reduce the number of injections, it was found that starting treatment at 7 wk of age with a twice daily dose of 5 mg/kg for about 8 d, followed by once daily injections of 5 mg/kg leptin, was an equally effective replacement treatment to produce fertility. Compared with protocol A, this protocol required approximately 20 injections and approximately 4.0 mg leptin to bring the female to conception. Thus, for the rest of the study, protocol B was used.

When leptin treatment was withdrawn from ob/ob females mated with +/+ males on either 0.5 or 3.5 dpc, only 8% of the females had a pregnancy that went to term. By contrast, significantly more of the ob/ob females mated to +/+ males had pregnancies that went to term when leptin treatment was withdrawn at 6.5 or 14.5 dpc or was given throughout pregnancy (75%; P < 0.002, by Fisher’s exact test; Table 1a and Fig. 1). When leptin was administered for 6.5 dpc or longer, 60% of all plugs were fertile, and 58% of the females conceived after the first plug. The 60% total plug rate achieved with leptin treatment compares well with the 72% observed in vehicle-treated +/+ control females mated with +/+ males (Table 2). Sterile matings can result from mating occurring outside estrus (12) or when insufficient intromissions take place (13). In this study it was not possible to distinguish between sterile matings and failure to conceive and/or implant, and it is likely that some of the matings where ob/ob females had leptin treatment withdrawn on either 0.5 or 3.5 dpc were sterile. The females in these two groups were all plugged only once even though the +/+ male was kept with the female throughout pregnancy. However, assuming that females treated with leptin for 0.5 and 3.5 dpc have the same proportion of success in conceiving at first plug as the females administered leptin for 6.5 dpc or longer, then withdrawing leptin at either 0.5 or 3.5 dpc significantly compromised these pregnancies from continuing to term (P < 0.05, by Fisher’s exact test; Table 1a).

View larger version (32K): [in this window] [in a new window]   Figure 1. Duration of leptin treatment and the associated pregnancy rates.

Regardless of sire (+/+ or leptin-treated ob/ob) and hence the genotype of the pups (either all ob/+ or all ob/ob, respectively), litter size and pup weights at birth were unaffected by withdrawing leptin at either 6.5 or 14.5 dpc (Table 1) and were no different from the outcome of wild-type matings (Table 3). The average (±SEM) litter size of wild-type matings (that is, vehicle-treated +/+ control females mated with +/+ males) was 6.4 ± 0.4 pups/litter (Table 3) compared with 6.6 ± 0.4 pups/litter produced by all leptin-treated ob/ob females (n = 43; Table 1). The average (±SEM) pup weight at birth in wild-type matings was 1.8 ± 0.1 g (Table 3) compared with 1.9 ± 0.1 g (Table 1) in ob/ob female offspring.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The restoration of hormone deficiency often produces spectacular results and none more so than treating the ob/ob mouse with recombinant murine leptin. Most, if not all, of the phenotypic signs of leptin deficiency are reversed or completely abolished, and this applies in particular to reproductive function. In this study two protocols were used. The initial approach (protocol A) was to identify ob/ob neonates by genotyping 10-d-old litters. Leptin treatment commenced at weaning (21 d of age) with doses designed to produce the same weight as in lean littermate controls. Genotyping is necessary because overt signs of obesity are not sufficiently reliable at weaning to indicate which animals are ob/ob. The second approach used to restore fertility with leptin (protocol B) started at 7 wk of age when ob/ob mice were clearly obese and at an age when they would have reached puberty. Both protocols were successful at restoring fertility in female and male ob/ob mice. However, in the results presented here, protocol B was used because it was realized that protocol A subjected the mice to an excessive number of leptin injections. Another welfare consideration was removing the need to genotype all the pups in a litter before weaning. Thus, protocol B was simpler, involved less stress on the animals, and allowed the establishment of a fixed dosing scheme that was used consistently throughout the study to produce fertile ob/ob mice. Male ob/ob mice for breeding purposes were treated by the same protocol and were test mated against lean females before being used for the experimental breeding program. The fertility of these males could be sustained by continuing treatment, but it proved more economical to recruit new males as and when required. Protocol A is recommended if one wants to study ob/ob mice that have developed normally after weaning rather than trying to reverse the effects of leptin deficiency in later life.

Having established a satisfactory protocol for producing fertile ob/ob females, it was decided to mate them with homozygotic lean (+/+) males so that all of the fetuses were heterozygotes (ob/+). This meant that the fetuses were capable of producing their own endogenous leptin and were more likely to develop normally and survive to term without necessarily having to rely on the exogenous leptin injected into the mothers. However, the proportion of successful full-term pregnancies when the males were also ob/ob was, if anything, better (82%) than that when using lean (+/+) males (75%). These pregnancy rates apply to those females receiving leptin throughout pregnancy, but pregnancies only went to term if leptin injections to the mother were continued for at least 6.5 dpc.

When leptin treatment was withdrawn at 0.5 or 3.5 dpc, only 1 of 13 pregnancies went to term, and we conclude that this was due to a failure of conception and/or implantation. This contrasts with the results reported by Mounzih et al. (10), who found that their ob/ob females would go to term when leptin treatment was withdrawn at 0.5 dpc. One explanation is that they used high doses of leptin to restore fertility in the ob/ob mice, that is, 50 mg/kg compared with the maintenance dose of 5 mg/kg used in the present study. The high dose used in the Mounzih et al. study (10) may have been due to the use of human rather than murine recombinant leptin; interestingly, the dose used was 2.5-fold greater than that used in their previous study (11). Another group (14) using murine leptin has been able to restore the fertility of ob/ob male mice with doses as low as 2.5 mg/kg, but these males were less successful in impregnating lean (ob/+) female mice (31% pregnant) than the 82% success rate achieved here with fertile ob/ob females. In the Mounzih et al. study (10) it is possible that there had been an accumulated reserve of leptin sufficient to last until approximately 5 dpc, when implantation normally occurs. Although recombinant human leptin has a half-life of 60 min in the circulation of the mouse (15), the turnover in tissues or the extent to which binding by the soluble binding protein (OB-Re) extends the half-life of leptin in the circulation is currently unknown. Another possible explanation for the difference is that the Mounzih et al. study (10) used the C57BL/6J strain of mouse, in contrast to the Aston strain used by us. A recent report has shown that a different genetic background can affect the degree of fertility in C57BL/6J ob/ob mice via the effect of modifier genes (16).

Leptin may be essential during the vital period between 0.5–6.5 dpc for a number of processes before and including implantation. Our experiments were only designed to identify critical periods of leptin requirement and not to investigate its actions. However, based on literature reports, speculations on its involvement in successful establishment of pregnancy can be made. For instance, leptin is present in the human oocyte (17), and although its function is unknown it is possible that it has a role in fertilization and the early cleavage stages of development. It is unlikely that leptin is required to ensure transport of the fertilized ova into the uterus, as Smithberg and Runner (18) showed that tubal transplantation of eggs occurred normally in ob/ob pregnant mice maintained on progesterone. Leptin may, however, be required for the induction of the estrogen surge (probably indirectly by stimulating LH release), which is essential for implantation (19). In addition, leptin is reported to have angiogenic activity (20) and may play an important role in the early establishment of a vascular system between blastocyst and uterus.

The results regarding the leptin requirement for conception and/or implantation differ from those reported by Mounzih et al. (10), but our results agree with their finding that, once established, leptin is not required for pregnancy to proceed to term. Furthermore, the litter size and weight of newborn pups were no different from those of litters produced by lean vehicle-treated control mice. Nevertheless, the newborn pups failed to survive unless the mother had been given leptin throughout pregnancy and continued after birth. Even if leptin treatment was continued up to 14.5 dpc and then restarted when the litter was born, the pups still failed to thrive. This indicates that there is a leptin requirement during pregnancy to ensure the development of the mammary glands and probably a continued requirement for lactation. Mounzih et al. (10) came to a similar conclusion, but in that study even pups delivered by continuously leptin-treated ob/ob females failed to survive beyond birth. However, these pups had not been given leptin during the suckling period. In a previous study by the same group (11) in which leptin was given continuously during pregnancy and lactation, most ob/ob mothers failed to suckle their young. It is possible that the higher leptin dose used by them may have prevented the normal deposition of fat in the breast pads, thus inhibiting normal mammary gland development. Mounzih et al. (10) also showed that the pups would survive if fostered onto a normal lactating mother. Experiments are now being undertaken to determine when and for how long during pregnancy leptin is required for mammary gland development and whether leptin is required throughout the entire suckling period. As well as leptin and the leptin receptor being expressed in mammary gland epithelia in wild-type mice (21, 22), leptin is secreted in the milk and may play an important role in modulating the metabolism of the neonate (23). Despite the above, it has to be remembered that the expression or appearance of leptin at different stages in fetal development does not necessarily mean that it has a function. In support of this concept, db/db and fa/fa neonates, lacking a functional leptin receptor, cannot respond to leptin in the milk, but they still appear to develop quite normally. Likewise, the fact that the ob/ob fetuses from the experimental matings apparently develop quite normally when leptin treatment is withdrawn at 6.5 dpc questions the role of the leptin that is expressed by normal fetuses in a variety of tissues, e.g. cartilage/bone, hair follicles, gut, and heart (6, 7). The phenotypic effect of leptin in these tissues is either undetectable or insignificant in normal early development.

In conclusion, there is a requirement for leptin in fertility, conception, and implantation, followed by a period during pregnancy and after parturition when leptin is required for proper mammary gland development and lactation. However, it would seem that both fetal and neonatal development can proceed normally in the absence of leptin or a functional leptin signaling pathway (e.g. db/db and fa/fa fetuses and neonates). This questions the role of the leptin expressed in normal fetuses and the leptin secreted in the mother’s milk.

	Footnotes

 
This work was supported by a grant from the Wellcome Trust.

Abbreviations: dpc, Days post coitum.

Received May 8, 2001.

Accepted for publication August 24, 2001.

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