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Biology of Reproduction 67, 1115-1124 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Impact of Growth Hormone Resistance on Female Reproductive Function: New Insights from Growth Hormone Receptor Knockout Mice1

Denise Zaczek2,a, James Hammondb, Lii Suenb, Serge Wandjib, Darlene Servicea, Andrzej Bartkea, Varadaraj Chandrashekara, Karen Coschiganoc, and John Kopchickc,d

a Department of Physiology, School of Medicine, Southern Illinois University, Carbondale, Illinois 62901-6512 b Department of Medicine and Cellular and Molecular Physiology, Division of Endocrinology, Diabetes, and Metabolism, Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033 c Edison Biotechnology Institute, Konneker Research Laboratories, Ohio University, Athens, Ohio 45701 d Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We examined multiple aspects of reproductive function in growth hormone receptor gene knockout (GHR-KO) and normal mice to clarify the role of growth hormone in female reproduction. In adult animals, estrous cycle duration was comparable in all mice housed individually but was significantly longer in group-housed GHR-KO females. Histological evaluation of ovaries of adult females at estrus showed that the numbers of preovulatory follicles and corpora lutea were significantly reduced in GHR-KO mice, as was the plasma estradiol level. The number of atretic preovulatory follicles was reduced in GHR gene-ablated animals. Although reverse transcription polymerase chain reaction analysis revealed reduced ovarian insulin-like growth factor I (IGF-I) mRNA expression in GHR-KO females, the expression of several steroidogenic enzyme mRNAs did not differ between groups. The numbers of active corpora lutea and uterine implantation sites were reduced in GHR-KO females at Day 7 of gestation. When young females were mated to normal males, latency to first mating and age of the female at first mating were significantly delayed in GHR-KO females, but maternal age at first conception was similar between groups. Significantly fewer virgin GHR-KO females exhibited pseudopregnancies when initially placed with vasectomized normal males than did normal female counterparts. Growth hormone resistance and IGF-I insufficiency negatively impacted 1) follicular development/ovulation rate, 2) sexual maturation, 3) production of and responsiveness to pheromonal signals, and 4) the ability of virgin females to respond to coitus by activation of luteal function. Although GHR-KO female mice are fertile, they exhibit quantitative deficits in various parameters of reproductive function.

growth hormone, ovary, ovulation, pregnancy, puberty


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although metabolic and growth-promoting actions of growth hormone (GH) and the insulin-like growth factors I and II (IGF-I and IGF-II) have long been recognized, reproductive roles for these hormones continue to emerge. GH and IGF-I and their receptors are widely distributed throughout the hypothalamic-pituitary-gonadal (HPG) axis, along with numerous binding proteins and proteases known to regulate GH/IGF bioavailability and action [1–4]. Putative reproductive roles for GH and IGF-I have been described at all levels of the HPG axis. Individually or in concert, these hormones have been implicated in the following aspects pertinent to female reproduction: 1) regulation of the timing of puberty onset [5–9] and the progression of sexual maturation [10–12], 2) regulation of GnRH and gonadotropin release from the hypothalamus and pituitary, respectively [13–19], and 3) modulation of ovarian follicular growth and development [20–24], steroidogenesis [25–31], and apoptosis [32–35]. In addition, GH and IGF-I influence numerous processes related to pregnancy, including oocyte maturation and fertilization [36–38], early embryo development [39], implantation [40, 41], fetal/placental growth and development [42–46], and litter size [47]. However, the underlying mechanisms responsible for these actions have yet to be clearly defined. To what extent specific effects are due to the actions of GH, IGF-I, or the combination of both factors is often unclear. Moreover, although the majority of circulating IGF-I derives from GH-stimulated production in the liver [48], extrahepatic IGF-I production is well documented and not uniformly GH dependent [49–51]. Thus, identifying the actions of GH-dependent versus GH-independent IGF-I is important for understanding the role of IGF-I in the control of reproduction.

Laron dwarf mice, developed by Zhou et al. [47] through targeted disruption of the GH receptor/binding protein gene, provide a unique model for clarifying the physiological roles of GH versus IGF-I and specifying the actions of GH-independent IGF-I. These GH receptor knockout (GHR-KO; -/-) mice lack functional GH receptors, are GH resistant, and exhibit proportionate dwarfism [47]. Lack of appropriate GH/IGF-I negative feedback in -/- GHR-KO mice results in significantly elevated levels of serum GH, to which the animals presumably do not respond. In contrast, circulating IGF-I levels are either significantly reduced or undetectable in these GHR knockouts, as compared with normal counterparts [19, 47, 52]. Although GHR-KO mice are fertile, in contrast to IGF-I knockouts (IGF-I-KO) [53], their reproductive performance is compromised. Significant reductions in litter size, increases in perinatal pup mortality, and advanced age at first conception in -/- x -/- crosses (compared with +/+ x +/+ and +/- x +/- pairings) were the primary reproductive deficits first described in GHR-KO mice [47]. Puberty is delayed in males [54] and females and can be advanced (at least in females) by administration of exogenous IGF-I [45]. Reductions in fetal size and weight with concomitant increases in placental size were evident in GHR-KO litters in late gestation and were influenced exclusively by maternal genotype, whereas reductions in litter size were influenced by both maternal and paternal genotypes [45]. Fertility in GHR-KO males is reduced [19], possibly because of altered pituitary and Leydig cell function [55].

The purpose of the present study was to define basic reproductive parameters in GHR-KO mice and to investigate maternal parameters that may contribute to the reduced litter size in these animals. Specifically, our objectives were to 1) determine the duration of the estrous cycle, 2) determine the mean number of preovulatory follicles (PF), corpora lutea (CL), and antral atretic follicles, and quantify plasma estradiol concentrations, 3) evaluate ovarian function at estrus using reverse transcription polymerase chain reaction analysis (RT-PCR) to assess ovarian mRNA expression for IGF-I and key steroidogenic enzymes, 4) assess the impact of GH resistance/IGF-I insufficiency on luteal function as manifested by frequency and duration of pseudopregnancy (psp) and serum progesterone levels and the numbers of active CL and embryo implantation sites in the uterus in early pregnancy, 5) determine whether the delay in first conception reported by Zhou et al. [47] is related to male or female factors, and 6) evaluate possible differences in social/pheromonal control of reproductive function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All experiments were performed in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction and with protocols approved by institutional committees. Animals were maintained under a 12L:12D photoperiod at 22 ± 2°C with ad libitum access to tap water and standard lab chow (Purina Formulab 5008 Rodent; El Mel Inc., Florissant, MO).

Effects of Genotype on Estrous Cyclicity and Ovarian Morphology

Female GHR-KO (n = 9) and normal (n = 10) mice 3–6 mo of age were individually housed. Vaginal smears were prepared daily (0800–0930 h), stained with 0.1% methylene blue dye, and used to determine the stage of the estrous cycle. All females were monitored by vaginal lavage for a minimum of two estrous cycles per animal. At predicted estrous phases, females were anesthetized with isoflurane for blood collection and then killed. Observation of the reproductive tract was used to verify accuracy of smear interpretation, using gross, edematous distension of the uterine horns or the presence of oocytes in the oviduct(s) as confirmation of estrus/ovulation. Data on estrous cycle duration (ECD) were analyzed for differences between populations using ANOVA and post hoc Fischer protected least significant difference (PLSD) tests, with significance at {alpha} = 0.05.

For morphological studies, ovaries were dissected and either fixed in 10% buffered formalin or frozen and processed for histological evaluation. Every 10th (hematoxylin and eosin stained) tissue section was analyzed by standard light microscopy (200x magnification) to determine the number of PF and CL per mouse. Frozen ovaries were sectioned on a cryostat, and 10-µm frozen sections (two/ovary) were processed for detection of apoptosis by the TUNEL procedure [56] using an immunochemical method ApopTag (Intergen, Purchase, NY) kit following the manufacturer's instructions. Data on number of PF, CL, and atretic follicles per mouse were analyzed for differences between populations using a t-test.

Effect of Genotype on Adult Ovarian Function and Circulating Estradiol

Young adult (2–6 mo old) normal and GHR-KO (n = 10 animals/group) female mice were individually caged. At estrus, plasma was collected under isoflurane anesthesia, females were killed, and ovaries were dissected and frozen at -60°C to -70°C for analysis of gene expression. Total RNA was isolated with a kit (Qiagen, Valencia, CA). Complementary DNA was generated by reverse transcription (RT) (Omniscript RT kit; Qiagen) and amplified by polymerase chain reaction (PCR) (PCR HotStar Taq DNA polymerase; Qiagen). Primers specific for glyceraldehyde phosphate dehydrogenase (GAPDH), a ubiquitously expressed gene, and one of the mRNAs of interest, including those for insulin-like growth factor I (IGF-I), {Delta}5-3ß-hydroxysteroid dehydrogenase (3ß-HSD), P-450 sidechain cleavage (P450scc), and aromatase, were employed in PCRs. For each reaction, the concentration of primers and number of cycles were optimized to assure linearity of the reaction for both the unknown and the control (GAPDH) gene. Primer sequences and optimized PCR conditions are listed in Table 1. Messenger RNA expression for the genes of interest was estimated from ethidium bromide-stained gels and normalized to GAPDH mRNA levels for each mouse. The number of samples (mice) analyzed (by t-test) represented all the samples in which RNA of sufficient quantity was available.


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  		TABLE 1. Optimized parameters for RT-PCR ovarian mRNA concentrations

Plasma samples from these females were stored at -60°C to -70°C until assayed for estradiol by RIA (Third Generation estradiol kit; Diagnostic Systems Laboratory, Webster, TX). The sensitivity of this assay was 5.5 ± 2 pg/ml and the (interassay) coefficient of variation (CV) was 12.8% at 10 pg/ml and 12.8% at 28 pg/ml.

Effect of Genotype on the Female Response to Sterile Mating

The proportion of females exhibiting normal psp in response to sterile mating and the duration of psp were determined for GHR-KO and normal females as an indication of luteal function. At the age of 3–6 mo, two GHR-KO or two normal females (n = 10 mice total per genotype) were placed with one vasectomized normal male of similar age and checked daily for vaginal plugs (between 0800 and 1030 h). The duration (days) of the intervals between consecutive plugs was recorded. Pseudopregnancy in mice lasts roughly 11 days [57–59]. Therefore, we considered intervals of 11 ± 3 days duration as potential psp events. Thirty-eight days after females were placed with vasectomized males in all cages where psp had been detected in both females (four of five cages containing normal females and three of five cages containing GHR-KO females), the paired females were transferred to new cages, an intact normal adult (3–6 mo old) male was added, and females were monitored to assess fertility parameters. The remaining cages were monitored until both females in each cage had demonstrated psp and had been similarly transferred to cages with intact males. One GHR-KO female died as a result of mating injuries. The remaining female was subsequently placed with an intact male in the absence of a second female. Chi-square analysis was used to determine whether the following parameters were genotype independent: 1) the proportion of females exhibiting only psp events and 2) the proportion of the total number of observed intervals that represented psp events. In addition, t-tests were used to determine differences between populations in 1) the mean duration of the individual intervals observed and 2) the mean latency (days) to first mating.

Effect of Genotype on Ovulation, Implantation Rate, and Serum Progesterone Concentrations at Day 7 Gestation

Females were placed with 3- to 6-mo-old intact normal males and monitored daily for vaginal plugs. At Day 7 of gestation (plug = Day 1), plasma was collected by cardiac puncture under isoflurane anesthesia, using 6% EDTA as anticoagulant, frozen at -60°C to -70°C, and subsequently analyzed for progesterone by RIA (Coat-A-Count Progesterone; Diagnostic Products Corp., Los Angeles, CA; assay sensitivity = 0.02 ng/ml) in a single assay according to manufacturer's instructions. The average CV for duplicate samples was 4.05% (range, 0.37–10.49%). Females were killed by cervical dislocation, and the number of active CL and uterine implantation sites was determined by gross macroscopic observation of the ovaries and uterine horns, respectively.

A t-test was used to determine significant differences between populations in the means for each parameter. Only data of those females confirmed pregnant by both elevated plasma progesterone concentration and the presence of at least one implantation site were used for statistical analysis. Data from four females (two normal and two GHR-KO) were omitted from statistical analysis because of nonconfirmation of pregnancies or insufficient plasma samples available for steroid assays. The Pearson correlation coefficient was used to determine linearity in the relationships of the number of CL and the number of uterine implantation sites or the serum progesterone concentration in early pregnancy (Day 7 of gestation).

Effect of Genotype on Age at First Conception in Females Mated to Normal Males

Prepubertal (24–39 days old) normal (n = 22) and GHR-KO (n = 13) female mice were placed (two females/cage) with one intact normal male (mean ± SEM age = 52.7 ± 2.6 days). To ensure that both GHR-KO and normal females would be similarly exposed to any male effects (e.g., delayed puberty, male infertility/subfertility) that might occur, all cages containing a GHR-KO female also contained a normal female. The remaining normal females were similarly placed (two females/cage except one cage that had only a single female) with an intact normal male. Although more normal than GHR-KO females were used, mice of both genotypes were distributed throughout the entire 24- to 39-day age span, and the mean age of females at pairing did not differ (P = 0.54) between groups. Overt size differences exist between normal males and females at this age. These size differences are very pronounced between normal males and GHR-KO females and presumably contribute to occasional injuries to the female during mating. Thus, to minimize mating-induced injuries in female mice, small virgin males were used for breeding purposes. Mice were checked daily (between 0800 and 1030 h) for vaginal plugs and parturition. The date of delivery and litter size were recorded. Two females (one GHR-KO and one normal) incurred mating-induced injuries and were prematurely eliminated from the study.

A t-test was used to determine whether differences existed between populations in 1) latency (days) to first mating (i.e., the occurrence of the first plug), 2) the age of the female at first mating, 3) maternal age at first conception, and 4) litter size. No significant differences were found in normal females housed with GHR-KO females versus those housed with other normal females, so data from all normal females were combined for final analyses. For two normal females, no plug was observed coincident with conception. Because gestation can last 19–20 days in these mice [45], only data from females with recorded plugs were used in statistical analyses of the age of the female at first mating and first conception and latency to first mating. Our experience has shown that although vaginal plugs are sometimes missed in normal females, they are rarely if ever missed in GHR-KO females, especially in very young or small GHR-KO females, where they occasionally had to be removed by the experimenter to avoid or alleviate bladder and/or bowel obstruction. Chi-square analysis was used to determine whether or not the following parameters were genotype independent: 1) the proportion of females that became pregnant and 2) the proportion of females that exhibited multiple plugs prior to first conception.

In reciprocal studies, prepubertal (35–36 days old) normal (n = 15) and GHR-KO (n = 6) males were placed (one male/cage) with two normal adult (3–6 mo old) females and monitored daily for date of delivery (DOD) of the first litter per cage. The age of attainment of fertility in males was determined by subtracting 19 days (mean length of gestation) from the age of the male at DOD for the first litter in his cage. A t-test was used to determine differences in age of attainment of fertility in males between groups.

Effects of Housing Condition and Genotype on Estrous Cyclicity

We investigated the possibility that social/pheromonal conditions might differentially affect ECD in GHR-KO and normal mice. A subset (n = 5 mice/genotype) of the mice used to determine the effect of genotype on ECD (previously described) were monitored by vaginal lavage for a period of 18 days under group-housed (5 animals/cage) conditions. The remaining mice in each genotype were housed individually during this period and also monitored by daily vaginal lavage. Group-housed females were subsequently switched to individual cages, and all mice except one were monitored for a minimum of two additional cycles. One female that was individually housed for the entire observation period manifested invariant 4-day cycles and was monitored for only one additional cycle. One initially group-housed GHR-KO female was omitted from the study because of continuously uninterpretable vaginal smears, and one initially group-housed normal female was omitted because of anestrus and hypogonadism detected at necropsy. Rarely, smears in other animals were uninterpretable. Only cycles in which every daily smear was interpretable were used for data analysis. Data on ECD were analyzed for differences between populations using ANOVA and PLSD test at alpha = 0.05, using observed cycles rather than individual mice as the experimental unit (n).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Genotype on Estrous Cyclicity

Mean ECD was roughly 5 days and did not differ between genotypes in female mice housed individually (normal: 4.75 ± 0.2 days; GHR-KO: 5.00 ± 0.17 days; P = 0.49).

Effects of Genotype on Ovarian Morphology and Function at Estrus

Genotype markedly influenced the mean number of PF and CL per mouse at estrus with both parameters significantly reduced in GHR-KO females compared with normal controls (PF: 42.9 ± 3.7 vs. 28.8 ± 3.5, P < 0.05; CL: 10.6 ± 1.2 vs. 4.5 ± 1.1, P < 0.005) (Fig. 1, A and B). The reduction in PF at estrus was not due to atresia of antral follicles, because the number of apoptotic antral follicles was reduced in GHR-KO animals (5.2 ± 1.6 vs. 0.6 ± 0.2, P < 0.02) (Fig. 1C). Nonetheless, the overall reduction in preovulatory follicles was sufficient to result in a significant reduction in serum estradiol levels in GHR-KO mice (8.2 ± 1.4 pg/ml vs. 2.7 ± 0.5 pg/ml, P < 0.003) (Fig. 1D).



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  		FIG. 1. Effect of genotype on the numbers of PF (A), CL (B), and atretic antral follicles per mouse (C) and on serum estradiol levels (D) in mice at estrus. Bars represent mean ± SEM. Atretic follicles were scored based on immunohistochemical staining for DNA fragmentation (n = 10 slides from five animals of each genotype). Data for A and B were collected from one group of mice and data for C and D were collected from a different group of mice.

To further address the nature and cause of the observed changes in ovarian function in GHR-KO animals, ovarian RNA from estrous animals was assayed for mRNA of IGF-I, steroidogenic enzymes, and GAPDH by semiquantitative RT-PCR. GHR-KO animals exhibited a 37% reduction in the expression of IGF-I mRNA (P < 0.003) (Table 2). In contrast, the concentration of mRNA for the three steroidogenic enzymes surveyed (aromatase, P450scc, and 3ß-HSD) did not differ significantly between groups (Table 2).


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  		TABLE 2. Effect of genotype on expression* of ovarian IGF-I and steroidogenic enzyme mRNAs

Effect of Genotype on the Luteal Response to Sterile Mating

GHR-KO females exhibited markedly different responses to sterile mating compared with normal controls. The proportion of females exhibiting only psp events (defined as intervals between consecutive plugs of 11 ± 3 days in duration) was lower in GHR-KO than in normal females, as was the proportion of the total observed intervals that represented psp events (Table 3). Moreover, latency to first mating was greater in GHR-KO than in normal females (2.2 ± 0.3 days vs. 5.8 ± 0.1 days) (Table 3). For those intervals that met our criteria for psp, mean duration did not differ (P = 0.70) between populations (normal: 10.2 ± 0.2 days; GHR-KO: 10.1 ± 0.4 days) and was consistent with previous reports on the duration of murine psp [57–59]. In two instances, females (both normal) exhibited intervals of >20 days between consecutive plugs. Collectively, interval data and daily notations on redness, swelling, and gaping of the external genitalia suggested that these longer intervals likely represented two consecutive psp events, with failed observation of vaginal plugs in between. These intervals were omitted from statistical analyses. For those intervals in GHR-KO mice that failed to meet our criteria of psp (11 ± 3 days), none exceeded the maximum duration. Rather, intervals not meeting the criteria of psp were in all cases shorter than 8 days duration, ranging from 2 to 7 days in length. Of the seven GHR-KO females that failed to demonstrate normal psp, five had multiple intervals of short duration (<8 days) during the period of observation. Two GHR-KO females exhibited a single interval too short in duration to constitute psp, followed by multiple psp events in each female. The remaining two GHR-KO females exhibited only psp events.


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  		TABLE 3. Effect of genotype on the response to sterile mating in adult female mice

When all the intervals were analyzed, the mean duration (days) of the first and second intervals was reduced in GHR-KO compared with normal females (interval 1: 10.2 ± 0.4 days vs. 6.1 ± 1.0 days, P < 0.005; interval 2: 10.1 ± 0.2 days vs. 6.7 ± 1.2 days, P < 0.01) (Fig. 2). In contrast, the mean duration of intervals 3 and 4 did not differ between populations. The percentage of females per interval that exhibited normal psp was consistently 100% for normal controls. In contrast, the percentage of GHR-KO females per interval exhibiting psp events progressively increased in consecutive intervals (Fig. 2).



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  		FIG. 2. Effect of genotype on the duration of the interval between consecutive plugs in adult female mice mated to normal vasectomized males. Intervals 1, 2, 3, and 4 represent the intervals between the first and second, second and third, third and fourth, and fourth and fifth consecutive plugs, respectively. Bars represent mean ± SEM. Values in parentheses above each bar indicate the percentage of all observed females that exhibited true psp during that interval. Values in the white box at the base of each bar represent the number (n) of females observed.

Effect of Genotype on CL Function and Uterine Implantation in Early Pregnancy

Genotype markedly affected the mean numbers of active CL and uterine implantation sites on Day 7 of gestation (Fig. 3). Compared with normal controls, GHR-KO females had fewer active CL (9.3 ± 0.6 vs. 4.9 ± 0.3) and uterine implantation sites (10.5 ± 0.7 vs. 5.8 ± 0.4) at Day 7 of gestation (Fig. 3). However, serum progesterone concentrations were not significantly different (P = 0.08) between populations (normal: 40.3 ± 4.7 ng/ml; GHR-KO: 29.9 ± 3.0 ng/ml) at this stage of pregnancy. There was a strong positive correlation (r = 0.97) between the mean number of CL and the mean number of uterine implantation sites and a weaker positive correlation between the mean number of CL and mean serum progesterone concentration (r = 0.42).



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  		FIG. 3. Effect of genotype on the numbers of active CL (A) and uterine blastocyst implantation sites (B) per mouse at Day 7 of gestation. Bars represent mean ± SEM.

Effect of Genotype on Age at First Conception in Females Mated to Normal Males

When young GHR-KO and normal females were mated to normal males, genotype did not significantly affect mean maternal age at first conception (P = 0.22), which occurred at roughly 6 wk in both populations (Table 4). Although the proportion of females that conceived did not differ significantly (P = 0.164) between groups, all normal females conceived, but only seven of nine GHR-KO females became pregnant. For both GHR-KO females that failed to conceive, the age of the female at first mating and the latency to first mating were greatly delayed compared with the remaining GHR-KO females (ages at first matings: 64 and 103 days; latencies to first matings: 39 and 79 days, respectively). Statistically, these two females were outliers, and their data were not included in the analyses of these parameters. However, genotype did influence a variety of other reproductive parameters (Table 4). Litter size was reduced for GHR-KO dams (P < 0.001). Perinatal pup mortality was 25% in litters of GHR-KO dams versus 3% in litters of normal dams, but this difference was not significant. Mean latency to first mating was delayed (P < 0.004) in GHR-KO females compared with control females, as was the mean age of the female at first mating (P < 0.0001) (Table 4). The age of the female at pairing was negatively correlated with latency to first mating only in GHR-KO females (normal: R = -0.24, P <= 0.31; GHR-KO: R = -0.71, P <= 0.04).


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  		TABLE 4. Effect of genotype on reproductive parameters in young female mice mated to intact normal males

The temporal pattern of matings also differed between groups. First matings of normal females were clustered and occurred within 15 days of introduction of the males. Mating peaked (5 of 20 females) in this population 4 days after males and females were paired, 1–3 females mated on all other days between Days 2 and 11 postpairing, and the final female mated on Day 15 postpairing. In contrast, no GHR-KO females mated within 4 days of being paired with males, and no peak in mating activity was observed. Rather, GHR-KO females mated for the first time sporadically, between 5 and 79 days after introduction of the males, and by 15 days postpairing only half of the GHR-KO females had mated. Multiple plugs were recorded for females in each group (30–40%), but this phenomenon was not genotype dependent (P = 0.89).

Attainment of fertility, defined as the earliest age at which GHR-KO and normal male mice impregnated normal adult females, was significantly delayed in GHR-KO animals (46.0 ± 1.4 days vs. 63.8 ± 2.9 days; P < 0.0001). The value for one normal male was a statistical outlier and was omitted from analysis. However, when statistical analysis was performed including that value, the difference remained highly significant (P < 0.0004). Additionally, one GHR-KO male failed to impregnate either of the females with whom he was caged for >90 days.

Effect of Genotype on Estrus Synchronization

Genotype, housing, and genotype x housing interactions all significantly affected mean ECD (P < 0.01, 0.0001, and 0.05, respectively) (Fig. 4). However, the effect of genotype was dependent upon housing conditions; ECD was not different (P = 0.49) in GHR-KO and normal females housed individually (Fig. 4). Estrous cycle duration was longer and more variable in group-housed females than in counterparts housed individually, and this effect was more pronounced in GHR-KO than in normal mice (group-housed: normal, 6.18 ± 0.66 days; GHR-KO, 8.43 ± 1.25 days; individually housed: normal, 4.75 ± 0.21 days; GHR-KO, 5.00 ± 0.17 days). Longer cycles in group-housed females were characterized by prolonged periods of diestrus. Moreover, atypical cellular associations were noted in the late metestrus/diestrus transition in all mice, characterized by abundant nucleated epithelial cells and mucus in the presence of numerous leukocytes.



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  		FIG. 4. Effect of genotype and housing on mean ECD in adult female mice housed individually (ih) or in groups (gh). Bars represent mean ± SEM. Different letter designations represent significant differences: b different from a and c at P < 0.04 and P < 0.005, respectively, and c different from a at P < 0.0001


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Absence of both systemic and local IGF-I in IGF-I-KO mice results in infertile females in whom the reproductive tract remains persistently juvenile and ovulation does not occur [53]. In contrast, GH resistance and the associated significant reductions in peripheral IGF-I compromise but do not abolish reproductive competence in GHR-KO mice [19, 45, 47]. In the current study, we further characterized the reproductive deficits seen in GHR-KO female mice. This study provides evidence that although puberty onset is delayed in GHR-KO animals, the age of attainment of fertility is similar in GHR-KO and normal females but is significantly delayed in GHR-KO males compared with normal males. Moreover, the current studies on postpubertal females suggest that decreased ovulation rate is the primary maternal variable associated with reduced litter size in GHR-KO animals, possibly related to decreased ovarian IGF-I mRNA expression. The possible altered responsiveness to male- and female-derived pheromonal stimuli in GHR-KO females may be responsible, at least in part, for a number of reproductive aberrations in these animals, including increased latency to first mating and prolonged ECD in group-housed females.

The ovarian studies showed a major decrease in the number of PF and in ovulation rate and a consequent decrease in the number of CL in adult GHR-KO animals. The number of apoptotic antral follicles was reduced in GHR-KO ovaries, suggesting that the observed reduction in follicle number possibly occurred at earlier stages of follicular development. As judged by the expression of mRNA for key steroidogenic enzymes, the follicles present seemed to have adequate machinery to produce steroids. However, the reduced number of competent follicles was inadequate to sustain normal estradiol levels or a normal ovulation rate. These deficits are likely central to the other changes in reproductive function, including reduction in number of implanted fetuses and litter size [45, 47].

The results of the current study shed light on the contribution of GH and IGF-I to ovarian function but fail to clearly resolve these effects. Ablation of GH signaling results in a dramatic decrease in circulating IGF-I [19, 47, 52] and a modest but significant decrease in ovarian IGF-I mRNA levels. These data are consistent with those of previous studies in rodents [60] and pigs [25], which have shown that ovarian IGF-I expression is GH responsive. However, at least in the pig, stimulation of IGF-I activity in the ovarian follicles by gonadotropins [61] and growth factors [62] seems equally important. These alternative pathways may account for the persistence of significant ovarian IGF-I expression in the GHR-KO genotype. These levels may be sufficient to allow development of a reduced number of functional PF. Our data show a decrease in atresia and no decrease in steroidogenic enzyme expression in mature GHR-KO follicles. Thus, the reduction in estradiol levels and ovulation rate could reflect a quantitative deficit in earlier follicle development. Researchers from another laboratory [63] also reported this defect in GHR-KO animals. This explanation would be congruent with observations in the IGF-I-KO mouse, in which follicular development ceases at the late preantral stage, possibly related to decreased FSH responsiveness [29]. The quantitative defect in PF (33% reduction) in the GHR-KO animals is comparable to the defect in ovarian IGF-I mRNA expression (37% reduction) and consistent with a central role of ovarian IGF-I in the reproductive phenotype of these animals. However, we cannot exclude a role for IGF-I-independent effects of GH on ovarian function from these studies nor can we eliminate the possibility of indirect effects of GH resistance on ovarian function in GHR-KO mice mediated by changes in neuroendocrine parameters. Several parameters of neuroendocrine function are significantly altered in GHR-KO animals [19, 55]. Circulating FSH levels are reduced in male GHR-KO mice [55], and ovarian FSH receptor (FSHR) gene expression, which was decreased in IGF-I-KO mice, was restored to control values by exogenous IGF-I treatment [29]. These data suggest that IGF-I plays a role in the development of ovarian FSHRs and FSH secretion. FSH is a major hormone required for folliculogenesis [64]. Therefore, it is tempting to speculate that the changes observed in ovarian function in GHR-KO mice in the present study were due to alteration in FSH secretion.

We have observed some heterogeneity in reproductive phenotype in GHR-KO females (unpublished results). Most females are fertile and exhibit the reproductive deficits noted in this study and previously [45, 47, 63]. However, a subset of GHR-KO females appear infertile, failing to conceive when mated multiple times with males of proven fertility (unpublished results). Although the underlying cause(s) of infertility in this subgroup remain unclear, neuroendocrine parameters may be more severely altered in these females or compensatory changes that allow for some level of reproductive competence in most GHR-KO females may be lacking or inadequate in the more profoundly affected subset of GHR-KO animals.

GH and IGF-I have both been implicated as regulators of the timing of puberty onset [5–9] and the progression of sexual maturation [10–12]. We previously reported that puberty onset, as assessed by age at vaginal introitus, was significantly delayed in GHR-KO females [45]. More recently, Keene et al. [54] demonstrated significant delay of puberty onset in male GHR-KO mice, as assessed by a variety of reproductive parameters. We mated young GHR-KO and normal females to normal males to determine to what extent delayed puberty in GHR-KO females contributed to the delayed age at first conception initially reported by Zhou et al. [47]. Results from the current study provide further evidence that puberty onset is delayed in GHR-KO females but suggest that age of attainment of fertility is not significantly different between GHR-KO and normal females. In contrast, attainment of fertility was significantly delayed in GHR-KO males compared with normal males. Normal males were capable of impregnating normal adult females at roughly 6 wk of age, consistent with the initial findings of Zhou et al. [47] for +/+ x +/+ and +/- x +/- pairs, whereas GHR-KO males were incapable of successful impregnation of normal females until roughly 9 wk of age. Thus, both puberty onset and attainment of fertility are delayed in male GHR-KO mice. Collectively, these findings suggest that delayed attainment of fertility in males, but not females, is likely the major factor contributing to the delayed age at first conception initially reported for GHR-KO mice [47].

Puberty can be advanced by about 10 days in normal prepubertal female mice by exposure to urine of adult male mice [65]. In the current study, although mean latency to first mating was about 1 wk for normal prepubertal females, peak mating occurred in these females 4 days after introduction of the male, suggesting that normal females were responsive to male pheromonal cues. In contrast, no prepubertal GHR-KO females mated within 4 days of introduction of the male, no peak in mating activity was observed, and the mean latency to first mating was delayed by roughly 10 days, compared with normal females. These findings raise the possibility that pheromonal responsiveness is impaired in GHR-KO females and that altered pheromonal sensitivity may prevent male-induced acceleration of female puberty. Alternatively or in addition, developmental delays may occur in GHR-KO females and underlie these reproductive aberrations, as suggested by the fact that latency to first mating was negatively correlated with age of the female at pairing only in GHR-KO animals. We cannot rule out the possibility that young normal females simply outcompeted young GHR-KO females in mating advantage. In the present study, estradiol levels at estrus were higher in normal adult females than in GHR-KO counterparts, which could render normal females more appealing and/or receptive to the male than GHR-KO females. However, increased latency to first mating in GHR-KO females was also observed in the current study, in which females were segregated by genotype, suggesting the existence of some factor(s) unrelated to competitive advantage of normal females that underlies these findings. The age of the female at first mating reported here likely represents the earliest age at which first ovulation and female receptivity occurred and suggests that these parameters, similar to vaginal introitus, are delayed in GHR-KO animals.

For virgin mice, the proportion of females that exhibited psp in response to sterile mating was significantly reduced in GHR-KO mice, compared with normal controls. When psp did not occur in GHR-KO females in response to mating, the interval between plugs was shorter (2 or 4–7 days in duration) than the minimum interval we defined as possible psp (8 days). Intervals of 4–7 days duration are consistent with normal cycles for these animals. We cannot explain the occurrence of intervals 2 days in duration, except to suggest that they possibly represent mating that occurred in the absence of female receptivity. Aberrant activation of luteal function in GHR-KO females could have resulted from the reduction in ovarian IGF-I and PF in adult animals. Alternatively, GHR-KO females may be unable to appropriately recognize or respond to coitus-induced neuronal signals essential for development of functional CL of pregnancy. However, with time and repeated mating, GHR-KO females were able to normalize their response and exhibited psp of comparable duration and with a frequency indistinguishable from that of normal controls. Irrespective of the mechanisms involved, these findings are reminiscent of reproductive functioning in human Laron-type dwarfs [66] and in a bovine model of immunoablation of GH secretion [67] in which pubertal mechanisms were more profoundly disturbed than reproduction in mature animals. Although the underlying mechanism(s) responsible for these effects is unclear, both GH and IGF-I influence neuronal development and plasticity [68, 69].

Latency to first mating in adult GHR-KO females mated with vasectomized normal males was significantly longer than that in normal controls. Female stimuli (pheromonal cues) may act in concert with male signals to induce estrous cycle synchronization [65]. Thus, aberrant sensitivity to pheromonal cues could impact the ability of signals derived from female or male conspecifics to influence puberty onset and adult estrous cyclicity or synchronization in GHR-KO mice. Generation of and responsivity to female pheromonal cues were examined indirectly in the group-housing experiment. ECD was more significantly increased by group housing in GHR-KO than in normal females, suggesting that GH resistance or IGF-I insufficiency alters sensitivity to female pheromonal signals in GHR-KO animals. These deficits could delay development of key neuronal pathways that permit normal pheromonal responsiveness or pheromone receptor expression in GHR-KO mice. The ability of GHR-KO males to induce estrous synchrony in normal females is also altered [70].

Such alteration in pheromonal function almost certainly has a major neuroendocrine locus. However, a role for ovarian deficits, especially in generation of pheromones, cannot be ignored. A better understanding of the relative importance and developmental sequence of neuroendocrine and gonadal effects of GH should result from further studies with this model. GH resistance or IGF-I insufficiency negatively impacted 1) follicular development and ovulation rate, 2) sexual maturation, 3) production of or responsiveness to pheromonal signals, and 4) the ability of virgin females to respond to coitus by activation of luteal function. Thus, although GHR-KO female mice are fertile, they exhibit quantitative deficits in various parameters of reproductive function.


   FOOTNOTES
 
1 This work was supported in part by NIH grants HD-37672 (A.B.) and HD-24565 (J.H.) and the Ohio State Eminent Scholar Program, including a gift from Milton and Lawrence Goll (J.K.). Back

2 Correspondence: Denise J. Zaczek, Department of Physiology, School of Medicine, Southern Illinois University, Life Science II, Room 250, Carbondale, IL 62901. FAX: 618 453 1517; dzaczek{at}siumed.edu Back

Received: 18 February 2002.

First decision: 4 March 2002.

Accepted: 15 May 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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