1 Reproductive Biology Research, Indian Institute of Chemical Biology, Jadavpur, Kolkata 700032, 2 Institute of Reproductive Medicine, Salt lake, Kolkata 700091 and 3 Division of Protein Engineering, Indian Institute of Chemical Biology, Jadavpur, Kolkata 700032, West Bengal, India
4 To whom correspondence should be addressed. e-mail: snkabir{at}iicb.res.in
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: galactosaemia/germ cell migration/premature ovarian failure/resistant ovary syndrome
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A number of reports document that women with galactosaemia eventually develop POF (Waggoner et al., 1990). Galactosaemia encompasses a broad spectrum of ovarian pathology. While some patients clearly demonstrated ovarian failure related to follicle depletion, others might exhibit normal appearing primordial follicles in ovarian biopsy but without growth and development (Fraser et al., 1986
). The pathophysiological attributes of POF in galactosaemic and other chromosomally competent women are likely to be different but possibly progress through similar clinical pathways (Twigg et al., 1996
). So, 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). It has been documented that the reproductive system, specifically the ovary, is one of the targets of galactosaemia. Galactose toxicity has been reported to induce follicular cell death (Gitzelmann and Steinmann, 1984
; Fraser et al., 1986
), which may be responsible for the premature depletion of follicular reserve. Since the females receive a finite follicle store during fetal life, the numbers of germ cells in the initial gonadal pool may also have an impact on the ovarian follicular reserve. The lower the number in the initial pool, the earlier the depletion of the follicular reserve. In the vertebrates, precursors of primordial germ cells (PGC) are thought to originate in the epiblast and then migrate to the developing gonad. Abnormalities in germ cell formation and/or migration could therefore be mechanisms favouring a deficient initial pool of germ cells in the ovary. We, therefore, reasoned that it should be of considerable interest to explore if deficient initial pool of follicular reserve could form the basis of premature depletion of follicles in POF, particularly those associated with galactosaemia.
Earlier reports have documented that feeding large amounts of galactose, which overpowers the normal capacity of the animal to metabolize this sugar, can make an animal model of galactose toxicity (Gibson, 1995; Segal, 1995
). In the rat, prenatal exposure to high-dose galactose has been shown to induce a galactosaemia-like condition and contribute to the POF components of human galactosaemia (Chen et al., 1981a
). The present study employs the same model to investigate the effect of galactosaemia on germ cell migration.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 Indian Institute of Chemical Biology. Sprague-Dawley rats maintained under good husbandry conditions of food and water ad libitum with a diurnal cycle of 12 h light: 12 h dark starting at 06.00 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 group; n = 21) or without (control group; n = 13) galactose from day 3 of conception continuing through parturition. Each day between gestation ages 1215 days at 16.00 h (± 15 min), a number of rats from both groups were laparotomized under light ether anaesthesia. Embryos from one uterine horn were dissected out and processed for histochemical localization of primordial germ cells. The embryos in the other uterine horn were maintained until parturition. Female pups of 1 or 2 days old (control: n = 30; gal-exposed: n = 39) were killed, and the livers were dissected out and biochemically assayed for crude liver epimerase activity.
Fixation and processing of embryos
Embryos were fixed in a solution of 6% mercuric chloride, 1% sodium acetate and 0.1% glutaraldehyde (Schulte and Spicer, 1983) for 18 h at room temperature. The embryos were dehydrated through graded alcohols and xylene, and embedded in composite paraffin blocks in sagittal orientation.
Histochemical localization of germ cells
Serial sagittal sections of 5 µm thickness were cut through the whole embryo. PGC were histochemically stained by the method described by Fazel et al. (1987). Briefly, sections were dewaxed and rehydrated through a descending ethanol series. Mercuric salts were removed by treatment with Lugol solution, the sections were washed in 0.1 mol/l phosphate-buffered saline (PBS), pH 7.2 for 2x5 min followed by incubation in a solution containing 1020 µg/ml DBA-HRP conjugate in 0.1 mol/l PBS, pH 7.2, containing 0.1 mmol/l CaCl2, MnCl2 and MgCl2 for 2 h at 4°C. On completion of the incubation, sections were thoroughly rinsed with PBS and incubated in a diaminobenzidine-hydrogen peroxide substrate medium (pH 7.0) for 15 min at room temperature (Graham and Karnovsky, 1966
). After thorough washing, the sections were finally counterstained with a 1% solution of Alcian blue in 3% acetic acid (pH 2.5).
Quantification of germ cells
The numbers of germ cells in each embryo were quantified by the method as described by Abercrombie (1946). The average diameter of
100 PGC in each group and on each day of examination was measured by an ocular micrometer at 400x magnification and calibrated with an object micrometer on the microscope plate. The total numbers of sections covering the region of expected localization of PGC on the specific days were noted. DBA reactive cells were counted in 1020 embryos on each day of examination with 1520 sections per embryo. Three persons, including one who was not associated with the study, did all the counting blindly. The average number of PGC in a section (P) was derived by the equation P = A x M / (L+ M), where A is the crude number of PGC seen in the section, M is the thickness (in µm) of the section, and L is the average diameter of PGC (in µm). The total number of PGC in each embryo (N) was derived by the equation N = P x n, where n is the number of sections obtained through the site of localization of PGC in individual embryos.
Biochemical estimation of liver UDP-Gal 4-epimerase activity
Livers from the female litters were excised out following autopsy. Within each group, three livers were pooled together. Protein contents of the crude liver extracts were estimated by the method of Lowry et al. (1951). Crude liver epimerase activity was assessed according to the coupled assay procedure of Darrow and Rodstrom (1968
) [Reaction volume: 1 ml containing 500 µl of 0.2 mol/l gly-gly buffer, pH 8.8; 0.07 µmol of UDP-Gal; 1 µmol of
-NAD, 400 units of UDP-Glc dehydrogenase and 100 µl of liver extract at 1 mg protein/ml concentration]. The formation of UDP-Glc from UDP-Gal was spectrophotometrically monitored at 340 nm for 5 min. Enzyme activity was expressed as Beckman unit/mg protein (1 Beckman unit = an increase in optical density by 0.001 per min under the conditions of the assay).
Statistics
The data were expressed as means ± SD, and n refers to the number of animals or determinations. Differences in germ cell numbers within and between groups were analysed by one-way ANOVA, and liver epimerase activity was analysed by Students t-test using Epistat package (Tracy L.Gustafson, 2011 Cap Rock Circle, Richardson, Texas, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The mean numbers of PGC per embryo are presented in Figure 2. In both groups a day-wise increase in the number of PGC was registered. However, when compared to controls, on each day of examination between days 1214, the numbers of PGC were significantly (P 0.0003) lower in the gal-exposed group. In 15-day old embryos DBA reactivity was evidenced at several additional sites including the luminal surface of the mesonephric duct and the developing hindgut. This accounted for >15% inter-observer variation in the counting results. The quantitative value of PGC in the day-15 embryo was not therefore taken into account. However, based on the qualitative assessment, it was almost certain that the germ cells that colonized the gonad in the gal-exposed group were fewer than those of the controls (Figure 1, plates 4A,B).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Females receive a finite pool of follicles during fetal development, and thereafter no additional follicles are formed. If the expenditure of follicles is regulated orderly, the finite follicle store serves the reproductive needs for life. After the onset of puberty, the expenditure of the follicles occurs in the forms of ovulation and atretic death. Since the loss of follicles by ovulation is relatively tiny, follicular atresia remains the dominant form of follicle depletion. So, premature depletion of ovarian follicular reserve may result from either an under-endowment of germ cells early in life leading to a deficient initial pool of germ cells, or an accelerated rate of atresia.
The pathogenesis of POF in galactosaemia is unclear. Possibly the disease is the manifestation of toxic effects of galactose and its metabolites. It has been documented that within the reproductive system, the ovary is the major target for the effects of galactosaemia. Galactose-1-phosphate accumulates in the ovary both pre- and postnatally in galactosaemic females, results in degradation of uridine nucleotides, and induces follicular damage and atresia (Gitzelmann and Steinmann, 1984; Fraser et al., 1986
). This effect may be causally linked with premature depletion of follicular reserve, the usual form of POF associated with galactosaemia. But the question of whether the ovarian pathology also has a prenatal origin has been the subject of several investigations. The available evidence, particularly that from animal models, suggests that both in-utero as well as postnatal insults may play a role in galactosaemia-associated gonadal pathology (Chen et al., 1981a
; Gibson, 1995
; Holton, 1995
). The present investigation tested the hypothesis that impaired germ cell migration leading to a deficient initial pool of oocytes could form the basis of premature depletion of follicular reserve in galactosaemia. Pregnant rats fed with a high-galactose diet were shown to have increased blood levels of galactose and galactose-1-phosphate in the fetus (Chen et al., 1984
), and produced a sequel of abnormalities in the litters that characterize human galactosaemia (Segal and Bernstein, 1963
; Spatz and Segal, 1983
). Chen et al. (1981a
) demonstrated that rats fed with a 50% galactose diet from day 3 of conception produced offspring with a deficiency of oocytes in the ovary that developed POF characteristics of galactosaemia. We thought it reasonable to adopt this experimental condition as a model for the present investigation. However, one minor modification was made in respect to dietary galactose content. During a pilot study, we observed that feeding with a 50% galactose diet was associated with a significant reduction in the body weight of the mother rats, and an increased incidence of in-utero fetal resorption and stillbirths. We therefore gradually minimized the galactose content of diets and considered 35% galactose as the optimum level since it exerted no adverse effect on the mothers body weight, and produced a mean litter size comparable with that of the controls. It is as yet unknown if galactose toxicity has any adverse influence on the process of implantation. To avoid over-exposure to galactose during the pre-implantation phase, the galactose-supplemented diet commenced from day 3 of conception and not from day 1.
In the vertebrates, the primordial germ cell precursors are thought to originate in the epiblast, form part of the extra-embryonic mesoderm, and then gradually migrate to the developing gonad. The precise cellular mechanisms involved in the guidance of germ cells to the genital ridge remain uncertain; however, some chemotaxis is clearly operational (Yeh and Adashi, 1999). Fazel et al. (1987
) demonstrated that a unique glycoconjugate with terminal GalNAc is selectively expressed only transiently on the PGC during the course of active migration. A potential role for this glycoconjugate in the process of migratory mechanism has therefore been envisioned (Alonso et al., 2002
).
Since GalNAc constitutes an integral component of the glycoconjugate on the surface of migrating PGC, and DBA has the binding specificity for GalNAc, we used DBA-HRP conjugate as a histochemical probe to identify and localize the presence of PGC in the migratory path. The present results demonstrate that the number of PGC at the day-specific sites on the path of migration is significantly lower and the number of cells that finally colonize the gonadal stroma of the embryos exposed to high galactose is reduced. These results indicate that exposure to high galactose adversely affects germ cell migration, ultimately resulting in the development of a gonad with a low initial pool of germ cells.
It may, however, be questioned if the effect was attributed to galactose supplementation directly and not to the reduction in food intake due to the altered composition and palatability of food. We did not measure food intakes directly, but as stated earlier, galactose in the diet was optimized at a level that did not affect the body weight gain of the mothers during the course of gestation. This may be considered as indirect proof that the galactose-fed rats were not underfed or malnourished. In fact, rats fed a high galactose diet eat more than control rats (Chen et al, 1984).
Two possibilities may be put forward to explain the presence of decreased numbers of germ cells on the path of migration: (i) the formation of germ cells was impaired (for some reasons not yet known) and therefore the numbers of PGC at the corresponding sites were proportionately lower, or (ii) the germ cells were produced optimally but they could not migrate because of impaired synthesis of GalNAc under the prevailing experimental condition. The latter possibility is based on the earlier suggestion that in galactosaemia the synthesis of oligosaccharides with terminal GalNAc may be impaired (Ornstein et al., 1992). Petry et al. (1991
) reported a reduction in synthesis of glycolipids that contain either galactose or GalNAc, and accumulation of precursors to these compounds in neonates with galactosaemia.
Galactose is normally metabolized to glucose through the co-ordinated activities of three major enzymes: galactokinase, galactose-1-phosphate uridyl transferase and UDP-Gal 4-epimerase. Epimerase is involved at multiple steps in the process of synthesis of GalNAc. We observed that epimerase activity is significantly decreased in the neonates exposed to high galactose. This observation provides indirect evidence to suggest that under our experimental conditions GalNAc synthesis is impaired, and this may have a causal link with impaired germ cell migration.
Liver epimerase activity was assessed in the litters born to the mothers who underwent mid-term laparotomy for collection of embryos from one uterine horn. The objective was to assess germ cell migration and epimerase activity in embryos and litters exposed to identical uterine milieu. It may therefore be argued that surgical stress during mid-pregnancy differentially caused the control and galactose-exposed pups to modulate the epimerase activity differently. This possibility, however, can be ruled out because in our pilot study we assessed germ cell migration and epimerase activity in embryos and litters collected from individual mother rats (data not shown). We observed that, irrespective of whether or not mid-gestation surgical intervention was involved, litters from gal-exposed mothers had significantly lower epimerase activity.
There is little insight into the mechanism by which surface glycoconjugates could influence directed mobility of PGC. However, a mechanism involving galactosyl transferase (Gal-Tase) has been implicated in the migration of neural crest cells. It has been proposed that transfer of galactose from UDP-Gal at the cell surface to a terminal N-acetylglucosamine (GlcNAc) in the basement membrane results in translocation of the neural crest cell surface in the process of their migration (Runyan et al., 1986). A similar mechanism may be operative in the migration of PGC. The components that would promote such activity on the surface of PGC or in their path are as yet unknown. However, it may be significant in this context to point out that intra-embryonic injection of
-lactalbumin (LA), a mammary gland-specific Ca2+ ion-binding protein, during the course of active migration of PGC significantly prevented the process of migration (unpublished data). LA is known to modify the catalysing effects of Gal-Tase (Riopelle and Dow, 1991
). In the absence of LA, the transferase catalyses transfer of galactose to either free or bound GlcNAc, while in its presence (in the mammary gland) the normal galactosyl acceptor specificity of Gal-Tase is altered from GlcNAc to glucose (because of a decreased Km for glucose) (Ramakrishnan, 2001
). A striking inhibition of germ cell migration was also observed when free GlcNAc was made available as an acceptor of galactose through intra-embryonic injection of GlcNAc (unpublished data). Taken together, these observations suggest that interference with transfer of galactose from UDP-Gal to GlcNAc may adversely affect germ cell migration. We postulate that the presence of fewer germ cells in the migratory path of the gal-exposed embryos may be attributed to interference with Gal-Tase activity. Earlier reports that galactosaemia interferes with the process of galactosylation (Jaeken et al., 1992
; Ornstein et al., 1992
; Prestoz et al., 1997
) further substantiate our postulation.
There is general agreement that ovarian failure in galactosaemia is the manifestation of toxic effects of galactose and its metabolites, but there exists no uniformity of opinion on whether the toxic effects are prenatal or postnatal events. Chen et al. (1981a) demonstrated that prenatal exposure to high galactose could reduce the oocyte numbers in the rat ovary, and they suggested that prenatal toxicity is responsible for ovarian dysfunction in galactosaemia (Chen et al., 1981b
). On the contrary, there are propositions that follicular destruction in galactosaemia is a postnatal rather than prenatal event (Kaufman et al., 1981
; Levy et al., 1984
). The present findings therefore are in agreement with the proposition made by Chen et al. (1981a,b).
It seems probable, however, that as suggested by Fraser et al. (1986
), galactose toxicity may induce follicular destruction at pre- as well as postnatal stages of life.
It may, however, be significant to note that under-endowment of germ cells occurs irrespective of the sex of the gonad, but unlike POF, testicular dysfunction is not a very frequent finding in human galactosaemia. Moreover, in a similar rat model for galactose toxicity, Chen et al. (1984) demonstrated no corresponding pre- or postnatal testicular toxicity in male rats. Perhaps this differential ovarian and testicular resistance to galactose toxicity resides in the differential mode of germ cell division in male and female gonads. In the females, oogonial mitotic divisions are ceased before or shortly after birth, and thereafter no follicular damage can be compensated by the regeneration of new germ cells, while spermatogonia has the ability to repopulate the testis by way of proliferation continuously throughout adult life.
In conclusion, the present investigation is perhaps the first study to document that under experimental galactosaemia-like conditions, germ cell migration is adversely affected and the gonad is endowed with a low initial pool of germ cells. This may form the basis of the early depletion of follicles leading to POF in women with galactosaemia.
![]() |
Acknowledgements |
---|
![]() |
FOOTNOTES |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alonso, E., Saez, F. J., Madrid, J. F. and Hernandez, F. (2002) GalNAc moieties in O-linked oligosaccharides of the primordial germ cells of Xenopus embryos. Histochem. Cell. Biol., 117, 345349.[CrossRef][ISI][Medline]
Chen, Y.T., Mattison, D.R. Feigenbaum, L., Fukui, H. and Schulman, J.D. (1981a) Reduction in oocyte number following prenatal exposure to a diet high in galactose. Science, 214, 11451147.[ISI][Medline]
Chen, Y.T., Mattison, D.R. and Schulman, J.D. (1981b) Hypogonadism and galactosemia. N. Engl. J. Med., 305, 464465.[Medline]
Chen, Y.T., Mattison, D.R., Bercu, B.B. and Schulman, J.D. (1984) Resistance of the male gonad to a high galactose diet. Pediatr. Res., 18, 345348.[Abstract]
Conway, G.S. (1997) Premature ovarian failure. Curr. Opin. Obstet. Gynecol., 9, 202206.[ISI][Medline]
Cramer, D.W., Harlow, B.L., Barbieri, R.L. and Ng, W.G. (1989) Galactose-1-phosphate uridyl transferase activity associated with age at menopause and reproductive history. Fertil. Steril., 51, 609615.[ISI][Medline]
Darrow, R.A. and Rodstrom, R. (1968) Purification and properties of uridine diphosphate galactose 4-epimerase from yeast. Biochemistry, 7, 16451654.[ISI][Medline]
Fazel, A.R., Schulte B.A., Thompson, R.P. and Spicer, S.S. (1987) Presence of a unique glycoconjugate on the surface of rat primordial germ cells during migration. Cell Differentiation, 21, 199211.[CrossRef][ISI][Medline]
Fraser, I.S., Russell, P., Greco, S. and Robertson, D.M. (1986) Resistant ovary syndrome and premature ovarian failure in young women with galactosaemia. Clin. Reprod. Fertil., 4, 133138.[Medline]
Gibson, J.B. (1995) Gonadal function in galactosemics and in galactose-intoxicated animals. Eur. J. Pediatr., 154 (Suppl. 2), S14-S20.
Gitzelmann, R. and Steinmann, B. (1984) Galactosemia: how does long-term treatment change the outcome? Enzyme, 32, 3746.[ISI][Medline]
Graham, R.C. Jr and Karnovsky, M.J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14, 291302.[ISI][Medline]
Holton, J.B. (1995) Effects of galactosemia in utero. Eur. J. Pediatr., 154 (Suppl. 1), S77-S81.
Jaeken, J., Kint, J. and Spaapen, L. (1992) Serum lysosomal enzyme abnormalities in galactosaemia. Lancet, 340, 14721473.[ISI][Medline]
Kaufman, F.R., Kogut, M.D., Donnell, G.N., Goebelsmann, U., March, C. and Koch, R. (1981) Hypergonadotropic hypogonadism in female patients with galactosemia. N. Engl. J. Med., 304, 994998.[Abstract]
Levy, H.L., Driscoll, S.G., Porensky, R.S. and Wender, D.F. (1984) Ovarian failure in galactosemia. N. Engl. J. Med., 310, 50.[ISI][Medline]
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265275.
Nelson, L.M., Kimzey, L.M., White, B. J. and Merriam, G.R. (1992) Gonadotropin suppression for the treatment of karyotypically normal spontaneous premature ovarian failure: a controlled trial. Fertil. Steril., 57, 5055.[ISI][Medline]
Ornstein, K.S., McGuire, E.J., Berry, G.T., Roth, S. and Segal, S. (1992) Abnormal galactosylation of complex carbohydrates in cultured fibroblasts from patients with galactose-1-phosphate uridyltransferase deficiency. Pediatr. Res., 31, 508511.[Abstract]
Petry, K., Greinix, H.T., Nudelman, E., Eisen, H., Hakomori, S., Levy, H.L. and Reichardt, J.K.V. (1991) Characterization of a novel biochemical abnormality in galactosemia: deficiency of glycolipids containing galactose or N-acetylgalactosamine and accumulation of precursors in brain and lymphocytes. Biochem. Med. Metab. Biol., 46, 93104.[ISI][Medline]
Prestoz, L.L.C., Couto, A.S., Shin, Y.S. and Petry, K.G. (1997) Altered follicle stimulating hormone isoforms in female galactosaemia patients. Eur. J. Pediatr., 156, 116120.[CrossRef][ISI][Medline]
Ramakrishnan, B., Shah, P.S. and Qasba, P.K. (2001) Alpha-Lactalbumin (LA) stimulates milk beta-1,4-galactosyltransferase I (beta 4Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine. Crystal structure of beta 4Gal-T1 x LA complex with UDP-Glc. J. Biol. Chem., 276, 3766537671.
Richardson, S.J. (1993) The biological basis of menopause. In Burger, H.G. (ed), Clinical endocrinology and metabolism: the menopause. Vol. 17, W B Saunders Company, London, UK, pp. 116.
Riopelle, R.J. and Dow, K.E. (1991) Neurite formation on laminin: effects of a galactosyltransferase on primary sensory neurons. Brain Res., 541, 265272.[CrossRef][ISI][Medline]
Runyan, R. B., Maxwell, G.D. and Shur, B. D. (1986) Evidence for a novel enzymatic mechanism of neural crest cell migration on extracellular glycoconjugate matrices. J. Cell Biol., 102, 432441.[Abstract]
Schulte, B.A. and Spicer, S.S. (1983) Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse. Histochem. J., 15, 12171238.[ISI][Medline]
Segal, S. (1995) In utero galactose intoxication in animals. Eur. J. Pediatr., 154 (Suppl. 2), S82-S86.
Segal, S. and Bernstein, H. (1963) Observations on cataract formation in the newborn offspring of rats fed a high-galactose diet. J. Pediatr., 62, 363370.
Spatz, M. and Segal, S. (1983) Transplacental galactose toxicity in rats. J. Pediatr., 67, 438446.
Twigg, S., Wallman, L. and McElduff, A. (1996) The resistant ovary syndrome in a patient with galactosemia: A clue to the natural history of ovarian failure. J. Clin. Endocrinol. Metab., 81, 13291331.[ISI][Medline]
Waggoner, D.D., Buist, N.R. and Donnell, G.N. (1990) Long-term prognosis in galactosaemia: results of a survey of 350 cases. J. Inherit. Metab. Dis., 13, 802818.[ISI][Medline]
Wentz, A.C. (1996) Resistant Ovary Syndrome. In Adashi, E.Y., Rock, J.A. and Rosenwaks, Z. (eds), Reproductive Endocrinology, Surgery, >and Technology. Vol. 2, Lippincott-Raven Publishers, Philadelphia, USA, pp. 13851392.
Yeh, J. and Adashi, E.Y. (1999) The ovarian life cycle. In Yen, S.S.C., Jaffe, R.B. and Barbieri, R.L. (eds), Reproductive Endocrinology: Physiology, Pathophysiology, and Clinical Management. 4th edn, W.B.Saunders Company, Philadelphia, USA, pp. 153190.
Zalitis, J., Uram, M., Bowser, A.M. and Feingold, D.S. (1972) UDP-glucose dehydrogenase from beef liver. In Ginsburg, V. (ed), Methods Enzymol. Vol. 28 (Part B), Academic Press, London, UK, pp. 430435.
Submitted on August 19, 2002; accepted on October 16, 2002.