1 Reproductive Biology Research, Indian Institute of Chemical Biology, Jadavpur, Kolkata 700032 and 2 Institute of Reproductive Medicine, Salt Lake, Kolkata 700091, West Bengal, India
3 To whom correspondence should be addressed. e-mail: snkabir{at}iicb.res.in
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Abstract |
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Key words: galactosaemia/galactose toxicity/premature ovarian failure/rat model/resistant ovary syndrome
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Introduction |
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A number of reports document that women with galactosaemia eventually develop POF (Kaufman et al., 1988; Waggoner et al., 1990
; Guerrero et al., 2000
). The pathophysiological attributes of POF in galactosaemic and other chromosomally competent women are likely to be different, but in both cases the disease possibly progresses through similar clinical pathways (Twigg et al., 1996
). Therefore, investigation of galactosaemic subjects may provide clues to some natural mechanisms underlying the pathogenesis of POF.
The causal link between galactosaemia and POF is not definitely known. There are suggestions, however, that premature depletion of ovarian follicular reserve is due to toxic effects of galactose and its metabolites (Cramer et al., 1989). Earlier reports have documented that feeding large amounts of galactose, which overpowers the normal capacity of the animal to metabolize this sugar, can produce an animal model of galactose toxicity (Gibson, 1995
; Segal, 1995
). Rodents exposed to high concentrations of galactose had been the models for the study of gonadal dysfunction in galactosaemia. Adult females and the offspring from dams exposed to galactose were reported to exhibit manifestations of diverse ovarian dysfunction that include delayed onset of puberty, decrease in numbers of ovarian follicles and follicular resistance to gonadotrophins. (Chen et al., 1981
; Meyer et al., 1992
). We have demonstrated recently that prenatal exposure to high galactose adversely affects oogonial migration and results in a low initial pool of germ cells in the developing gonads (Bandyopadhyay et al., 2003
). Parallels can be drawn between these specific gonadal dysfunctions and POF or ROS components of human galactosaemia. With the foregoing considerations in mind, we reasoned that it should be of considerable interest to explore if experimental galactose toxicity can serve as a model for the investigations on POF/ROS. The present investigation demonstrates a composite picture of the ovarian dysfunctions under experimental galactose toxicity in rats, and evaluates its suitability as a model to unveil the pathophysiological mechanisms underlying the development of the disease.
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Materials and methods |
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Animals
The experiments were performed in accordance with the guidelines formulated by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Culture, India, with approval from the Animal Ethics Committee of the Indian Institute of Chemical Biology. SpragueDawley rats maintained under good husbandry conditions of food and water ad libitum with a diurnal cycle of 12 h light and 12 h dark starting at 0600 h were raised in our institute animal house. Adult female rats were mated with proven fertile males of the same strain, and the presence of sperm in the vaginal lavage was assigned as day 1 of gestation. They were fed standard food pellets supplemented with (gal-exposed; n = 38) or without (control; n = 24) galactose from day 3 of conception, continuing through weaning of the litters on postnatal day (PND) 21 (PND0 being the day of birth) when the female litters were separated and maintained separately, and employed for the following investigations.
Estimation of galactose and galactose-1-phosphate
Blood levels of galactose and galactose-1-phosphate were measured at weaning (PND21) (control, n = 12; gal-exposed, n = 15) as well as adult age (PND70/71) (control, n = 7; gal-exposed, n = 11). Blood was collected by direct cardiac puncture; sera were procured after centrifugation and assayed for simultaneous determination of galactose and galactose-1-phosphate by a colorimetric microassay method. The assay protocol was a modification of that described by Diepenbrock et al. (1992), wherein measured volumes of sera and standards were used instead of dried blood spots and standards. Measurement was done in the form of galactose alone or with that released from galactose-1-phosphate following co-incubation with alkaline phosphatase and galactose dehydrogenase. The concentrations were expressed as mmol/l.
Assessment of growth rate
If not otherwise mentioned, body weights (BWs) of the litters were recorded every 5th day since birth, and continued throughout the course of the experiments.
Assessment of onset of puberty
The onset of puberty was marked by the time of vaginal opening (VO). From PND35 onward, the rats (control, n = 30; gal-exposed, n = 57) were checked daily for VO. On the day of VO, age and BW of the respective rats were recorded, and during the subsequent course of investigations estrous cyclicity was evaluated by vaginal cytology.
Assessment of ovarian follicular reserve
Ovaries of the 55- to 60-day-old animals were dissected out immediately after sacrifice, trimmed of fat, weighed and immerse-fixed in Bouins fixative for 24 h followed by paraffin embedding and subsequent processing for routine histopathological evaluation under light microscopy.
Assessment of pituitary gonadal functions
Either at weaning on PND21 or on PND42/43, rats were euthenized. Blood was collected by direct cardiac puncture, and serum was separated and stored at 70°C for subsequent determination of LH, FSH and E2.
Serum levels of LH and FSH were measured by a double-antibody radioimmunoassay method. Pure rat LH (NIDDK-rLH-I-9) and FSH (NIDDK-rFSH-I-9) were iodinated with carrier-free 125I using chloramine T according to the method of Greenwood et al. (1963). The antisera to LH and FSH were NIDDK-anti-rLH-S-11 and NIDDK-anti-rFSH-S-11, respectively. The sensitivities of the assays were 0.15 ng/ml for LH and 0.5 ng/ml for FSH. All samples were assayed in a single batch at two concentrations, and each in duplicate. The intra-assay coefficients of variations were <8%. The LH and FSH values were expressed in terms of the reference standards, NIDDK-rat-LH-RP-3 and NIDDK-rat-FSH-RP-2.
Serum levels of E2 were measured by fully automated chemiluminescence assay system, ACS 180 (Chiron Diagnostics Corp., East Walpole, MA). The sensitivity of the assay was 10 pg/ml and the intra-assay coefficient of variation was 5.6%.
Assessment of follicular response to gonadotrophins
Between PND25 and PND30, control (n = 25) and gal-exposed (n = 35) rats were subjected to induction of the superovulatory response by exogenous gonadotrophins. They had an s.c. injection of 30 IU of PMSG at 0 h. A subset of animals from each group (control, n = 10; gal-exposed, n = 14) were sacrificed at 50 h when blood was collected, and serum was procured and stored frozen at 70°C until assessed for E2 levels. The remaining animals had an s.c. injection of 50 IU of HCG at 52 h followed by autopsy at 72 h when the presence of oocytes in the oviduct was assessed and counted. In another set of control (n = 15) and gal-exposed rats (n = 23), superovulation was effected after induction of pituitary desensitization. The rats had an s.c. injection of 100 µg of GnRH agonist per day in two equal doses for 10 days, and were then subjected to the induction of superovulation by PMSGHCG. The ovulatory response was assessed with respect to both numbers of rats exhibiting the presence of ovulated oocytes and the numbers of oocytes ovulated per rat stimulated.
Statistics
The data were expressed as mean ± SEM, and n refers to the number of animals or determinations. Differences between the groups were analysed by one-way ANOVA, Students t-test and 2 tests, as applicable, using the Epistat package (Tracy L.Gustafson, 2011 Cap Rock Circle, Richardson, TX 75080).
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Results |
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Growth rate
BWs of the litters at birth and subsequent growth rates are presented in Figure 1. At birth, the BWs of the pups were not statistically different, although the gal-exposed pups had relatively lower weight. Though both groups exhibited a steady rise in their BW, the rate of growth was significantly lower in the study population throughout the period of study. The difference in the growth rates reached the maximum level of significance during PND3040 when the control group exhibited a pre-pubertal growth spurt.
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Ovarian histology
Ovarian sections of control rats (Figure 3A) showed the presence of follicles at different stages of maturation and the corpus luteum (CL). In contrast, the study population exhibited diverse architecture of the ovaries (Figure 3B and C). Reduced ovarian mass and absence of graafian follicle and CL were generalized findings. However, many ovaries were deficient in small follicles (Figure 3B), while the others had an infantile appearance with a normal complement of follicles but with growth and differentiation arrested at the early pre-antral stage (Figure 3C). The presence of a large number of pyknotic granulosa cells, arranged in a single symmetric ring adjacent to the basement membrane and offering the structure of a string of beads, marked the preponderance of atretic cells (Figure 3B).
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Discussion |
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Women with galactosaemia eventually develop POF (Kaufman et al., 1988; Waggoner et al., 1990
; Guerrero et al., 2000
). It has been proposed that the pathogenesis of POF in galactosaemic and other women possibly progresses through similar clinical pathways (Twigg et al., 1996
). Thus, investigations on galactosaemic subjects are likely to unravel some natural mechanisms that may underlie the development of POF (Twigg et al., 1996
).
There is good evidence that galactose readily crosses the placenta (Segal, 1995). Administration of galactose to rats during pregnancy produces a sequela of abnormalities in the litters which are characteristic of human galactosaemia (Segal and Bernstein, 1963
; Spatz and Segal, 1983
). Thus the rodents provide an excellent model system for galactose toxicity, which is easy to acquire, maintain and modulate. We thought it reasonable to evaluate the potentiality of this experimental condition as the proposed model for the present investigation.
Increased levels of galactose and its metabolites are thought to be responsible for most of the toxic manifestations of galactosaemia. To assess the magnitude of galactose toxicity under the prevailing experimental condition with our treatment regimen, we measured the levels of galactose and galactose-1-phosphate in the circulation. As expected, both were found to be significantly elevated at weaning as well as adult age.
Little information is available on pre-pubertal growth in chromosomally competent women who subsequently developed POF. However, retrospective analyses of large series of galactosaemic POF women showed that many affected women had severely delayed growth during childhood and early adolescence, although normal growth and development was also very common (Waggoner et al., 1990). In a rat model, Chen et al. (1984
) demonstrated that galactose toxicity had an adverse impact on intra-uterine embryonic growth. The present investigation, however, documents only a smaller and insignificant reduction of mean litter weight at birth, although subsequent growth rates were significantly retarded. This differential growth rate among different galactosaemic women or between the experimental models may be attributed to the difference in magnitude of galactose toxicity. We had observed earlier that feeding rats with a 50% galactose diet, the dosage at which Chen et al. (1981
) observed a significantly reduced birth weight of the pups, was associated with a significant reduction in the BW of the dams, and an increased incidence of in-utero fetal resorption and stillbirths. We therefore gradually minimized the galactose content of the diets and considered 35% galactose as the optimum level since it exerted no adverse effect on the mothers BW and mean litter size was comparable with that of the controls.
With regard to the onset of puberty, there are a number of pertinent observations. Delayed puberty is a frequent finding in women with galactosaemia (Fraser et al., 1986). Late onset of puberty under experimental galactose toxicity has been reported previously (Chen et al., 1981
). The present results also document that the timing of VO, a reproductive tract response to ovarian steroids and one of the key indices for puberty in rodents, was significantly delayed under galactose toxicity. The onset of puberty involves a chain of events including increased production of sex steroids by the gonads in response to increased secretion of gonadotrophins from the anterior pituitary. This is driven by a spurt in the secretion of GnRH following activation of the hypothalamus. Exactly when the puberty-specific increase in the cascade of hormones begins largely depends on body size, rather than chronological age (Foster and Nagatani, 1999
). Since the gal-exposed litters had a retarded growth rate, it may be argued that delayed onset of puberty was not a direct effect of galactose toxicity, but rather attributed to delayed growth rate. To resolve the issue, we evaluated VO separately in relation to age and BW. We observed that 63% rats of the study group did not exhibit VO despite their BWs being within the 95% CI of the controls at the time of VO. This observation suggests that galactose toxicity per se is responsible for delayed puberty. Whatever the mechanism, delayed puberty generally results from an ultimate lag in activation of one or more of the factors determining the maturation of the hypothalamo-pituitaryovarian (HPO) axis (Rosenfield, 1996
). Observation of elevated gonadotrophins with low circulating levels of E2 in the study group, however, discounts the possibility of inactivation at the hypothalamo-pituitary level and points to the ovary as the possible primary target of galactose toxicity.
In immature female rats, FSH secretion remains high until PND18 and decreases thereafter (Thyssen and Libertun, 1996). A subsequent rise in FSH level occurs at the time of VO. This sequential drop and rise of FSH secretion is likely to be of phenomenal importance in the process of onset of puberty. Extended central nervous system (CNS) restraint on GnRH release is considered an attractive hypothesis for delayed puberty (Rosenfield, 1996
), but prolongation of high FSH by inhibition of polyamine synthesis also delays the onset of puberty independently of body mass (Thyssen and Libertun, 1996
). A drop in FSH during PND18 has been suggested to be a steroid-independent event of cerebral origin, while the rise of gonadotrophins that immediately precedes VO is brought about by a pre-pubertal gradual rise in estrogen secretion that acts at the CNSpituitary level to trigger a pre-ovulatory pro-estrous-like surge of gonadotrophins (Ojeda et al., 1976
). We demonstrated that the gal-exposed litters had elevated FSH levels both at weaning and at the stipulated peri-pubertal phase; and E2 levels remained significantly low throughout. It is therefore tempting to speculate that the absence of stimuli in the form of E2 perhaps blunts the puberty-specific sensitization of the CNSpituitary unit.
Food restriction during late gestation and lactation in rats has been reported to have negative impacts on the HPO axis, and to prohibit follicular maturation and delay the onset of puberty (Leonhardt et al., 2003). That the rats maintained on our dietary regimen were not underfed or malnourished has been substantiated in our earlier report (Bandyopadhyay et al., 2003
). Moreover, we also maintained the protein content of the galactose diet at 13.65% so that the basic protein requirement (12%) for pregnant rats is met. Therefore, the possibility of any implications of reduced food intake and consequent malnutrition on delayed puberty and related endocrinopathies under galactose toxicity can be ruled out.
Galactosaemia encompasses a broad spectrum of ovarian pathology. While some patients clearly demonstrate ovarian failure related to follicle depletion, others have primordial follicles appearing normal in ovarian biopsy but lacking growth and development (Fraser et al., 1986). In order to typify the ovarian effects of galactose toxicity, we investigated the ovarian follicular reserve and also evaluated its response to gonadotrophins. Chen et al. (1981
) reported that prenatal galactose toxicity led to a significant reduction in the number of small follicles. Our histopathological examination, however, did not reveal such a generalized effect. While many rats suffered from deficient follicular reserve, others exhibited apparently normal follicular complement, though they were unresponsive to exogenous gonadotrophins. Thus the basic tenets of the diverse phenotypes of POF are also conserved in experimental galactose toxicity.
The size of a follicular complement at any point of reproductive life represents the balance between the numbers of follicles endowed in the initial ovarian pool and those expended since then. A deficient follicular reserve may therefore ensue from an under-endowment of follicles in the ovarian pool and/or an accelerated rate of atresiathe major form of follicular expenditure. Within the reproductive system, the ovary is considered to be one of the major targets of galactose metabolites that have detrimental effects on the germ cells, granulosa cells, stromal cells and thecal cells (Kaufman et al., 1987, 1989; Gibson, 1995
). Fraser et al. (1986
) demonstrated that ovarian accumulation of galactose-1-phosphate led to degradation of uridine nucleotides and induced follicular atresia. In the present study, we observed a general preponderance of atretic secondary follicles in the gal-exposed group. On the other hand, we have demonstrated recently that galactose toxicity adversely affected germ cell migration leading to ovary development with a low initial pool of germ cells (Bandyopadhyay et al., 2003
). We therefore suggest that an increased rate of atresia as well as a deficient initial ovarian pool of germ cells are implicated in the process of premature depletion of follicles under galactose toxicity.
The state of hypergonadotrophic hypoestrogenism in the presence of an apparently normal complement of follicles under galactose toxicity attests to the state of follicular refractoriness to gonadotrophins. There are, however, suggestions that in galactosaemic women, some aberrant forms of gonadotrophins are produced that are biologically inactive (Prestoz et al, 1997). In order to replace such putative defective gonadotrophins, we induced superovulation in rats by exogenous gonadotrophins. Ovarian response, expressed in terms of the number of rats exhibiting ovulation or the number of oocytes ovulated per rat, was significantly poorer in the study group. It was not surprising that ovaries with deficient follicles responded poorly to gonadotrophins. However, it was significant that post-autopsy histological analysis of the gonadotrophin-exposed ovaries showed them to possess an apparently normal complement of unstimulated follicles (data not shown). If reduced biopotency of the endogenous gonadotrophins were the sole factor responsible for follicular refractoriness, these follicles should have responded to the exogenous gonadotrophins. In order to look for additional factor(s), we carried out the induction of superovulation after pituitary desensitization with chronic administration of GnRH agonist. The objective was to assess the ovarian response to exogenous gonadotrophins without any intervening effects of endogenous gonadotrophins. A significant increase in ovarian response in the GnRH-downregulated group raises the possibility that endogenous gonadotrophins had also exerted anti-gonadotrophic activity, presumably at the receptor level. It may, however, be relevant in this context that in galactosaemic patients, some neutral isoforms of FSH are produced that are partially deficient in galactose and sialic acid in the carbohydrate side chains (Prestoz et al., 1997
). These altered forms of FSH are characterized by their higher binding affinity for FSH receptors but reduced ability to activate the second messenger system. By virtue of this property, these isoforms of FSH may act as anti-hormones and produce antagonistic effects at the receptor level (Sairam, 1989
).
Earlier workers have proposed some genetically engineered mouse models for POF. Pellas et al. (1991) described an insertional mutation in mouse that caused a deficiency of primordial germ cells reaching the genital ridge during embryonic development. Dong et al. (1996
) effected targeted deletion using stem cell technology to produce growth differentiation factor-9 (GDF-9)-deficient mice that had unhindered formation of primordial and primary follicles but suffered from follicular development beyond the primary one-layer stage. Both of these experimental conditions could potentially be valuable animal model for human POF. However, if the diverse phenotypes of POF are taken into consideration, each model has its own limitations. A germ cell-deficient mouse may be reminiscent of only the phenotype of the follicle depletion type of POF, while a GDF-9-deficient mouse has the potential to be a model for the follicle dysfunction type of POF. However, galactose toxicity is unique in that it encompasses both spectra of the disease.
Finally, diverse aetiology and multifaceted phenotypes of POF limit the potential of a single model in encompassing the full spectrum of the pathogenesis of the disease. Importantly, however, in view of the foregoing, we are left with the conclusion that experimental galactose toxicity has the credentials to serve as a model at least to explore some of the basic tenets of POF/ROS irrespective of aetiology.
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Acknowledgements |
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Submitted on October 7, 2003; resubmitted on May 21, 2003; accepted on June 30, 2003.