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a Department of Obstetrics & Gynecology and
b Department of Human and Molecular Genetics, Baylor Collegeof Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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x Agt-/+ 
. There were 4 breeding pairs per scheme. Breedings were time mated. Mice and litters were weighed daily. Southern blotting was used for genotyping. We found that Agt-/- breeding pairs had fewer litters (2 [range 1–2] vs. 4 [range 3–5]; P = 0.01), fewer pups per litter (4 [range 1–7] vs. 6 [range 1–10]; P = 0.006), and longer interpregnancy intervals (43 days [range 31–44] vs. 35.5 days [range 22–58]; P = 0.04) compared to wild-type controls. The ratio of postcoital plugs to subsequent litters was 4.0 and 1.2 for Agt-/- and Agt+/+ breedings, respectively (P = 0.03). Median maternal weights during all trimesters of pregnancy were significantly lower for Agt-deficient mice compared to wild-type controls. Among Agt-/+ x Agt-/+ breedings, the proportions of Agt+/+ (n = 17), Agt-/+ (n = 38), and Agt-/- (n = 4) offspring differed significantly from the expected 1:2:1 Mendelian inheritance pattern (P = 0.03). Neonatal survival among the offspring derived from the Agt-/- x Agt-/- breeding scheme was significantly reduced (P = 0.001). We conclude that Agt deficiency is associated with an in utero lethal effect, decreased fertility, and impaired neonatal survival.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Besides the well-described role of the renin-angiotensin system (RAS) in vasopressor, electrolyte, and fluid homeostasis [5], a role for the RAS in embryonal development has been suggested. Various components of the RAS, i.e., AGT, ATII, and ATII receptors (AT1 and AT2), are expressed in human embryos from 3 wk to 40 wk gestation [6]. Inhibition of RAS components results in structural and functional abnormalities of the kidneys [7]. Production of ATII has been linked to heart, brain, liver, and adrenal gland development [6,8,9]. Several mechanisms with respect to RAS-mediated organ development have been proposed, among them increased transcription of protooncogenes, potentiation of growth factor effects, stimulation of extracellular matrix production, and direct growth-promoting effects [10,11]. However, the role of the RAS in embryonal development has to be interpreted with caution, since mice deficient in the AT-converting enzyme (ACE) encoding gene (Ace) are viable and develop morphologically normal organs, with exception of the kidneys [12].
A functional role for the RAS in reproduction and fertility has also been suggested [2]. This is based on the finding that local tissue-specific components of the RAS are found at all levels of the hypothalamo-pituitary-gonadal axis [3]. Others have demonstrated that the luteotropic hormone (LH) peak of the menstrual cycle is preceded by a surge of ATII production in the arcuate nucleus of the hypothalamus [4]. Moreover, sex steroids are known to stimulate hepatic and renal AGT production [2], and ATII has been demonstrated to stimulate the ciliary beat frequency of epithelial cells in the fallopian tubes [13]. A testis-specific isoform of ACE has been described, found only in developing spermatids and mature sperm [14]. Mice deficient in testis-specific ACE have sperm with reduced fertilization potential, defective oviduct transport, and defective binding to zonae pellucidae [14]. Some studies [15–17], but not others [12,18], suggest that testis-specific RAS is essential for sperm motility and capacitation. Experimental studies with respect to the influence of the RAS on reproductive outcome have yielded controversial results. Some studies, using targeted mutagenesis of the murine Ace gene [12,19], demonstrated reduced male fertility. Rescue of this phenotype was achieved with transgenic expression of Ace driven by the sperm-specific human pgk2 promoter [20]. Others, using ACE inhibitors [18,21] and ATII receptor antagonists [22], found no effects on male reproductive performance. Adding to the level of controversy with respect to reproductive effects of the RAS, pharmacologic inhibition of ACE yielded no effect on gonadotropin profiles and other menstrual cycle characteristics among hypertensive women [23]. Although deficiency in the murine AGT gene (Agt) has been noted to be compatible with male fertility [24], the influence of Agt on reproductive fitness has not been investigated. The purpose of this study was to investigate the efficiency of reproduction of male and female mice with 0 (Agt-/-), 1 (Agt-/+), and 2 (Agt+/+) copies of the Agt gene.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice, genetically engineered to have 0 (Agt-/-), 1 (Agt-/+), and 2 (Agt+/+) copies of the Agt gene on a mixed background, were kindly provided by Dr. O. Smithies (University of North Carolina, Chapel Hill, NC) [25]. Four breeding schemes were established: 1) Agt-/- x Agt-/-, 2) Agt-/+ x Agt-/+, 3) Agt+/+ x Agt+/+, and 4) Agt+/+ x Agt-/+ . Each mouse in the breeding scheme was considered sexually mature, based on an age of 10 wk. There were 4 breeding pairs per scheme. All breeding pairs were housed in separate cages except for the period during which timed mating took place. The duration of the mating period was 2 h per day (0900–1100 h). On an hourly basis, vaginal ostia were gently checked for a copulatory plug using an appropriately sized blunt glass rod. Identification of a copulatory plug defined the first day of gestation. After delivery of a litter, mating was restarted on Day 2 postpartum. Average pup weights were evaluated by daily weighing of litters in aggregate divided by the number of pups at that given day. Animal husbandry practices followed guidelines established by the Animal Care Committee of Baylor College of Medicine. Animals were subjected to daily 12-h alternating periods of light (0700–1900 h) and dark cycles within a humidity (63%)- and temperature (72–74°F)-controlled environment. Food and water were provided ad libitum. Female mice were weighed daily. Weaning and genotyping of pups were performed on postnatal day 21. Data for each breeding pair were collected over a 4-mo interval beginning with the establishment of the breeding pair of interest. The study was completed over an 8-mo period.
Testicles and prostate glands of 4 Agt-/-, 4 Agt-/+, and 4 Agt+/+ males and uteri, fallopian tubes, and ovaries of 4 Agt-/-, 4 Agt-/+, and 4 Agt+/+ females were harvested and histologically (hematoxylin and eosin stained) examined at the end of the observation period.
Genotyping
A genomic DNA fragment was amplified by the polymerase chain reaction to obtain a 725-base pair (bp) probe used for genotyping. The oligonucleotide primer pair used to generate this probe was derived from the published sequence of mouse AGT exon 2 (GenBank, accession No. AF045884, accession date 03/01/1998). The sequence of the forward 20-mer primer was 5'-GTA TAC ATC CAC CCC TTC CA-3'. This primer started at position 985 within exon 2. The reverse 22-mer primer sequence was 5'-GGA AGT GAA CGT AGG TGT TGA-3' starting at position 1710 within exon 2. Polymerase chain reaction products were resolved on a 1.5% low-melting agarose gel. The 725-bp fragment was recovered from the gel and purified.
The strategy used for Southern blot genotyping employed data related to the construction of the Agt knockout mice [25]. Briefly, mouse genomic DNA for genotyping was obtained from proteinase K-digested tails using phenol-chloroform extraction. The extracted DNA was stored at 4°C until analyzed. Genomic DNA was digested using the restriction enzyme SacI. Equal amounts of DNA (15 µg) were electrophoresed on a 1% agarose gel, denatured in 0.4 M NaOH for 45 min, and transferred overnight onto nucleic acid transfer membranes (Amersham, Piscataway, NJ) using 10-strength SSC (0.75 M sodium chloride and 0.075 M sodium citrate, pH 7.0; Fisher Scientific, Fairlawn, NJ). The membranes were prehybridized for 6 h at 64°C in a hybridization buffer containing 0.5 M sodium phosphate (Fisher Scientific), 1 mM EDTA (J.T. Baker, Phillipsburg, NJ), 7% SDS (Boehringer-Mannheim, Indianapolis, IN), 10% PEG 8000 (Fisher), 1 g/ml BSA (fraction V; Sigma Chemical Co., St. Louis, MO), and 100 µg/ml denatured salmon sperm DNA (Sigma). Southern blots of genomic DNA were then hybridized at high stringency with a 725-bp mouse AGT cDNA probe, which had been labeled using random primers ([
-32P]dCTP) as described by Shine et al. [26]. After hybridization for 18 h at 64°C, membranes were washed sequentially at 64°C for 20 min with 5-strength SSC and 0.1% SDS (Sigma), 2-strength SSC and 0.1% SDS, and 1-strength SSC and 0.1% SDS. The membranes were air dried, wrapped in plastic wrap, exposed to an autoradiography film (Amersham) for 48 h, and scored by visual inspection of autorads. In wild-type mice the SacI restriction fragment length polymorphism has a size of 6.0 kilobases (kb) [27]. In mice with targeted mutagenesis of exon 2 of Agt, the size of the SacI restriction fragment length polymorphism is 7.5 kb due to insertion of the targeting construct. This difference was used to distinguish between zero-copy and wild-type alleles. Figure 1 illustrates Southern blots for Agt-/-, Agt+/+, and Agt-/+ mice.
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Outcome Measures
Fetal loss For the purpose of this study, criteria for fetal loss included the following: 1) maternal weight gain on Day 13 after identification of a vaginal plug noted to have increased 20% over that seen on Day 1 of gestation; 2) subsequent continuous decline in maternal weight during Days 14–16 of pregnancy; 3) no subsequent litter.
Embryonic loss For the purpose of this study, embryonic loss, i.e., embryonic waste, was defined as first-trimester abortion or resorption.
Interpregnancy intervals Interpregnancy intervals were defined as number of days between consecutive litters.
Plug:litter ratio The ratio between copulatory plugs and subsequent litters, i.e., the plug:litter ratio, was used to determine the median number of plugs necessary for a successful pregnancy.
Statistics
The statistical software Sigma Stat version 2.0 (Jandel Scientific, San Rafael, CA) was used for statistical analysis. Survival probabilities of pups were calculated by the product limit method of Kaplan and Meier [28]. Differences between groups were tested using the log-rank test (Minitab, State College, PA). The results were analyzed for the endpoint of overall survival. Comparisons between unpaired groups were made using the Kruskal-Wallis test. Dunn's test was used for multiple comparisons of Agt-/+ and Agt-/- mice with a control group, i.e., Agt+/+ mice. Student's t-test was used to compare plug:litter ratios, and chi-square analysis was used to compare actual versus expected genotypes among offspring. Significance was assumed at = 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Table 1 shows that median maternal weights of Agt-/- females were significantly lower on days of gestation 7, 14, and 21, but not on Day 1, compared to those of Agt+/+ females. Figure 2 depicts maternal weights throughout all days of pregnancy among Agt-/-, Agt-/+, and Agt+/+ females. The median time interval from the setup of the breedings to the first litter and the median duration of pregnancy were not different among the three genotypes studied (Table 1).
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Agt-/- breedings showed a significantly reduced median number of litters compared to Agt+/+ breedings (2 [range 1–2] vs. 4 [range 3–5], respectively; P = 0.01). Of note, Agt-/+ breedings also showed a significantly reduced number of litters (2, range 1–3; P = 0.01) versus Agt+/+ breedings. The absolute number of litters observed among the breeding schemes were 7, 9, and 14 for Agt-/-, Agt-/+, and Agt+/+ breedings, respectively.
The median number of pups born per litter was 6 (range 1–10) among Agt+/+ breedings. Among Agt-/- breedings, this number was significantly lower (4, range 1–7; P = 0.006). Agt-/+ breedings showed no difference with respect to number of pups per litter (7, range 5–9; P = 0.2) when compared to Agt+/+ breedings.
Interpregnancy intervals were significantly longer for Agt-/- breedings (43, range 31–44) compared to Agt+/+ breedings (35.5, range 22–58; P = 0.04). No significant difference was observed between Agt-/+ breedings (41, range 28–99) and Agt+/+ breedings (P = 0.4). Analysis of covariance showed that differences between Agt+/+ and Agt-/- breedings with respect to the number of pups per litter and the duration of interpregnancy intervals were independent of the number of pregnancies (P = 0.01 and P = 0.04, respectively). Evidence for fetal loss was observed in 1, 2, and 1 cases among Agt-/-, Agt-/+, and Agt+/+ females, respectively. No case of maternal death was observed.
The median number of days between delivery of a litter and the next copulatory plug observed was 22 (range 3–39) for Agt-/- breedings, 20 (range 1–30) for Agt-/+ breedings, and 18 (range 1–39) for Agt+/+ breedings. These differences were not statistically significant (P = 0.5 for Agt-/- vs. Agt+/+ and P = 0.6 for Agt-/+ vs. Agt+/+). In contrast, the median plug:litter ratio was 3.5 (range 2.5–5), 3.0 (range 1.7–7), and 1.3 (range 1–1.7) for Agt-/-, Agt-/+, and Agt+/+ breedings, respectively (P = 0.01 for Agt-/- vs. Agt+/+ and P = 0.02 for Agt-/+ vs. Agt+/+). Among the four breeding pairs combining an Agt+/+ and an Agt-/+ , lower median litter numbers (2.5 [range 2–3] vs. 4 [range 3–5]; P = 0.03) and a higher plug:litter ratio (1.9 vs. 1.3; P = 0.05) compared to wild-type controls were observed.
Fetal Outcomes of Agt-/-, Agt-/+, and Agt+/+ Breedings
Twenty-one-day overall survival rates were 12%, 45%, and 77% among the offspring of Agt-/-, Agt-/+, and Agt+/+ breedings, respectively. A Kaplan-Meier survival analysis showed that overall survival was significantly reduced for Agt-/- versus Agt+/+ offspring (P = 0.001, Fig. 3 and for Agt-/+ versus Agt+/+ offspring (P = 0.002, Fig. 3). Pup weights of Agt-/-, Agt-/+, and Agt+/+ offspring throughout postnatal days 1–21 are shown in Figure 4. Among Agt-/+ breedings, the proportions of Agt-/- (n = 4), Agt-/+ (n = 38), and Agt+/+ offspring (n = 17) differed significantly from the expected 1:2:1 Mendelian inheritance pattern (P = 0.03). Offspring of the four breeding pairs combining an Agt+/+ and an Agt-/+ showed no deviation from the expected genotype distribution (P = 0.3), and the median number of pups per litter did not significantly differ from that observed among wild-type breedings (5 [range 3–9] vs. 6 [range 1–10], respectively; P = 0.4). The 21-day survival rate of pups did not differ significantly from that observed among wild-type breedings (70% vs. 77%, respectively; P = 0.1).
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Histologic Evaluation of Reproductive Organs
Testicles and prostate glands of 4 Agt+/+, 4 Agt-/+, and 4 Agt-/- males showed no histological evidence of abnormal structure or morphology. Uteri, fallopian tubes, and ovaries of 4 Agt+/+, 4 Agt-/+, and 4 Agt-/- females also showed no histological evidence of abnormal structure or morphology.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results are in accordance with previously reported data indicating a functional role of the RAS in reproduction [12,19,20]. We found homozygous Agt-deficient breeding pairs to produce fewer litters and smaller litter sizes compared to wild-type controls. These observations suggest that either male, female, or both male and female Agt-deficient mice have reduced fertility. Previous studies reporting on ACE-deficient mice showed female reproduction to be unaffected [12,19,20]. Our data show that Agt deficiency does not significantly affect maternal reproductive outcome measures such as time to first litter and duration of pregnancy. Taken together, this evidence suggests that AGT is not critically involved in the functioning of the female hypothalamo-hypophyseal-gonadal axis.
Ace knockout mice have been reported to display reduced male fertility [12,19,20]. We found that Agt-deficient breeding pairs had a higher plug:litter ratio than wild-type breeding pairs. Sperms of Agt-deficient males may thus be less likely to fertilize ova than those of wild-type males. A male contribution to reduced fertility is also suggested by the fact that breeding pairs with Agt heterozygous males and wild-type females exhibited lower litter numbers and a higher plug:litter ratio than wild-type breeding pairs. Taken together, these observations indicate that the reduced fertility observed among Agt-deficient mice may be attributed to a male component. It is a shortcoming of this study that all possible breeding combinations were not investigated to further separate maternal and paternal genetic effects. However, the data presented indicate that a lack of Agt reduces male fertility. Follow-up studies with respect to reproductive functions among Agt-/- males, e.g., sperm function and motility, are justified.
Timed mating took place in the morning hours. Since mice are nocturnal animals, we cannot rule out the possibility that the mating activity may have been reduced in the morning hours compared to nocturnal conditions. This has to be taken into account in interpretation of the results of this study.
Maternal weight gain patterns, as shown in Figure 2, and median maternal weights during pregnancy were significantly lower for Agt-deficient mice than for wild-type controls. Agt-deficient mice also delivered fewer pups per litter. The inheritance pattern among offspring of Agt-/+ x Agt-/+ breedings showed a skewed distribution of Agt genotypes. We attribute these differences to an in utero lethal effect caused by Agt deficiency. Of note, weight differences were observed throughout all trimesters. Additionally, we found no evidence for increased fetal loss (i.e., second- or third-trimester loss) among Agt-deficient breedings. This suggests an early onset, i.e., embryonic waste, as the cause for increased prenatal mortality of Agt-/- mice. A higher plug:litter ratio, as observed among Agt-deficient breedings, may also be secondary to increased embryonic wastage, as excess plugs that do not lead to a successful litter may indicate resorbed pregnancies. Since expression of RAS components has been described in the embryonic development of the kidney, liver, heart, and adrenal gland [6,8,19], it may be speculated that Agt deficiency contributes to embryonic loss by interfering with proper organ development. It could be argued that Agt deficiency, being associated with lower blood pressure [24], might lead to impaired placental blood flow and, consequently, to intrauterine growth restriction and higher perinatal mortality. In our experiment, all breedings were time mated. The duration of pregnancy was therefore known for all breedings. We observed no difference in pregnancy duration between Agt genotypes. Thus, prematurity as a possible cause of or contributing factor to lower pup weights and increased perinatal mortality can be ruled out.
As to the influence of Agt on neonatal outcome, few data have been reported suggesting impaired postnatal survival and reduced size among Agt-deficient pups [24,29]. In our series, Kaplan-Meier survival analysis for liveborn pups of all genotypes showed a significantly lower 21-day overall survival rate for offspring of Agt-/- and Agt-/+ breedings compared to wild-type controls. Agt-deficient pups in our series were smaller and showed less weight gain than wild-type controls. No catch-up growth was seen as would be expected in case of intrauterine growth restriction due to placental insufficiency. Lack of Agt is associated with hypotension [27]. Reduced growth potential and viability of Agt-deficient pups may therefore be attributed to impaired nutritional support.
We conclude that Agt plays a functional role in murine reproduction. Our data suggest that Agt deficiency is associated with reduced male fertility but does not interfere with female onset of sexual maturity, pregnancy duration, and the postpartum resetting of the hypothalamo-hypophyseal-gonadal axis. Our data suggest increased embryonic waste due to Agt deficiency. Furthermore, neonatal outcome measures suggest absence of Agt results in increased postnatal mortality.
Various mutations and polymorphisms of the human AGT gene (AGT) have been described, e.g., M235T and L11V [30,31]. These markers may be promising candidates for assessing the genetic contribution to infertility in a clinical setting. A correlation between Agt deficiency and early embryonic waste suggests that variants of AGT may also be an important determinant in recurrent first-trimester pregnancy loss.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by Erwin-Schroedinger-Auslandsstipendium J1592MED with funding by the Fonds Zur Foerderung der Wissenschaftlichen Forschung (to C.T.); National Institute of Child Health and Human Development (NICHD) Grant R03-HD-34667-01 (to A.R.G.); Methodist Hospital Foundation Grant (to A.R.G.). 
2 Correspondence: Anthony R. Gregg, Department of Gynecology and Obstetrics, Baylor College of Medicine 6550 Fannin-Suite 901, Houston, TX 77030. Fax: 713 798 6956; agregg{at}bcm.tmc.edu
Accepted: September 24, 1999.
Received: June 18, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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