- Email: [email protected]
or Lactation Negatively Affects Follicular Ovarian Development and Steroidogenesis in the Prepubertal Rat Offspring
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Archives of Medical Research
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ORIGINAL ARTICLE
Maternal Protein Restriction During Pregnancy and/or Lactation Negatively Affects Follicular Ovarian Development and Steroidogenesis in the Prepubertal Rat Offspring Q3
Carolina Guzman,a Rocıo Garcıa-Becerra,b Marco Antonio Aguilar-Medina,b Isabel Mendez,c Horacio Merchant-Larios,d and Elena Zambranob
a
HIPAM, Unidad de Medicina Experimental, Hospital General de Mexico, Facultad de Medicina, Universidad Nacional Autonoma de Mexico (UNAM), Mexico, D.F., Mexico b Departamento de Biologıa de la Reproduccion, Instituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiran, Mexico, D.F., Mexico c Instituto de Neurobiologıa, UNAM, Campus UNAM-Juriquilla, Mexico d Departamento de Biologıa Celular y Fisiologıa, Instituto de Investigaciones Biomedicas, UNAM, Mexico, D.F., Mexico Received for publication November 12, 2013; accepted March 14, 2014 (ARCMED-D-13-00640).
Background and Aims. Maternal protein restriction during rat pregnancy and lactation is associated with alterations in reproductive function of female offspring including delayed onset of puberty, decreased fertility and premature reproductive aging. These alterations may be related to ovarian prepubertal development, distribution of follicle populations and their steroidogenic capacities. We undertook this study to evaluate the ovarian function of prepubertal female offspring exposed to maternal protein restriction during pregnancy and/or lactation. Methods. Adult female Wistar rats were fed a control (C-20% casein diet) or restricted isocaloric diet (R-10% casein) during pregnancy—first letter—and lactation—second letter, to form four groups, CC, RR, CR, RC. Ovaries were collected from 21-day-old female offspring. Preantral and antral follicles were quantified and mRNA expression of key genes involved in follicular development and steroidogenesis (gonadotropin receptors, StAR, P450scc and P450 aromatase) was evaluated. Serum gonadotropin levels were measured. Results. Significantly decreased numbers of preantral and antral follicles were observed in CR and RC ovaries compared with CC. LH levels were lower and FSH higher in CR pups. mRNA expression of LH receptor (LH-R) was decreased in RR in comparison with the other groups. CR and RC expressed higher StAR, RC increased and RR decreased P450scc, whereas RR and CR decreased aromatase expression in comparison with CC. Conclusions. Maternal protein restriction influences prepubertal ovarian follicular number and steroidogenic function in the rat offspring, although RR and CR nutritional schemes have similar outcomes, the mechanisms affecting ovarian function are at different levels of the hypothalamic-pituitary-ovarian axis. Ó 2014 IMSS. Published by Elsevier Inc. Key Words: Maternal protein restriction, Ovary, Prepubertal development, Developmental programming.
Introduction
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Addressed reprint requests to: Dr. Elena Zambrano, Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiran, Vasco de Quiroga 15, Tlalpan 14000, Mexico, D.F. Mexico; E-mail: [email protected]
Programming of reproductive function has been demonstrated in response to exposure to a suboptimal environment during fetal and postnatal development (1e7). Maternal
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protein restriction during rat pregnancy alters key components of maternal steroid function increasing fetal exposure to estradiol, testosterone, progesterone and corticosterone (2,8). In the female offspring, onset of puberty and estrous cycles are delayed (1,8), fertility rates decreased (3,8), and reproductive lifespan shortened (8). Nutritional challenges during early life have been associated with altered pubertal development. Maternal protein restriction during lactation in the rat delays vaginal opening and first estrous, independently of maternal nutrition during pregnancy (8). In contrast, females fed high fat from weaning have advanced onset of puberty (4,9). In humans, migration has allowed researchers to identify the developmental effects of environmental changes during early life. For example, Bangladeshi girls raised in the UK have higher salivary progesterone levels if they migrate earlier than menarche. When migration occurs at a later age, they show lower progesterone salivary levels and later pubertal maturation (10). Sexual maturation and fertility depend on an adequate ovarian function, which is based on the availability of a pool of follicles for recruitment and ovulation (11) as well as an appropriate hormonal environment. In rats, follicular development begins in neonatal life when follicular structures are established in numbers that will progressively decrease through the reproductive lifespan (11,12). Ovarian follicles contain an oocyte surrounded by follicular cells. The numbers of follicular cell layers depends on the stage of follicular development (11,13,14). Follicles are classified according to the layers of granulosa cells as well as presence or absence of the antral cavity (15). In the rat, well-developed antral follicles are present by post natal day 15e17 (12). Ovarian steroidogenesis starts before puberty. Prepubertal estradiol levels regulate the establishment of pulsatile LH secretion (16). Both gonadotropins, LH and FSH, regulate expression of their receptors and the enzymes involved in sex steroid synthesis (17). Antral follicles express LH and FSH receptors and are capable of aromatization (14). Most of the prepubertal antral follicles undergo atresia, only a few reach the preovulatory stage under gonadotropin stimulation (11,14,18,19). FSH prevents programmed demise in early antral follicles (14). We hypothesized that assessment of prepubertal ovarian development and function could provide an early indicator of programming of sexual development due by maternal protein restriction during pregnancy and/or lactation. We assessed the number of the ovarian preantral and antral follicles in ovaries of 21-day-old offspring exposed to maternal protein restriction during pregnancy and/or lactation. Expression of ovarian proteins involved in follicular development and steroidogenesis (LH receptor (LH-R), FSH receptor (FSH-R), steroidogenic acute regulator (StAR), P450 side chain cleavage (P450scc) and P450 aromatase), as well as LH and FSH serum levels were measured.
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180 181 Care of Animals 182 183 Details of protein restriction and generation of pups have 184 been published previously (2). Briefly, 3-month-old virgin 185 female albino Wistar rats (240e260 g) were obtained from 186 the Instituto Nacional de Ciencias Medicas y Nutrici on Sal187 vador Zubiran (Mexico City, Mexico) and maintained under 188 controlled lighting and temperature (lights on 07:00 h to 189 19:00 h at 22e23 C). All procedures were approved by 190 the Institutional Animal Experimentation Ethics Committee 191 of the Instituto Nacional de Ciencias Medicas y Nutrici on 192 Salvador Zubiran, Mexico City and are in accordance with 193 Mexican and international laws for laboratory animal care. 194 Rats were mated overnight with proven male breeders 195 and the day spermatozoa were present in the vaginal smear 196 was designated day of conception, day 0. Only rats 197 becoming pregnant within 5 days of mating were studied. 198 Pregnant rats were transferred to individual cages and allo199 cated at random to one of two groups fed either 20% casein 200 (control dieteC) or 10% casein isocaloric diet (restricted 201 dieteR) (2). Food was provided as large flat biscuits re202 tained behind a grill through which rats nibbled the food. 203 Food and water were available ad libitum. 204 Vaginal delivery occurred spontaneously in the early 205 daylight hours between 09:00 and 12:00 h on post206 conceptual day 22. Day of delivery was considered as day 207 0 of postnatal life. Timing of delivery, litter size and pup 208 weight were recorded at birth. Anogenital distance was as209 sessed to determine pup sex using our previous criteria (8). 210 Sex was judged according to whether anogenital distance 211 was lower (female) or larger (male) than 2.5 mm. To ensure 212 homogeneity of study subjects, litters over 14 pups were not 213 included. Litters of 12e14 pups were adjusted to 12 pups for 214 each dam, maintaining as close to a 1:1 sex ratio as possible. 215 Four groups were established CC, RR, CR and RC. First let216 ter refers to maternal diet in pregnancy and second diet in lac217 tationeC control, R restricted. We report data on female 218 offspring only. 219 At 21 days of postnatal life, one female pup per litter was 220 rapidly euthanized by decapitation by experienced personnel trained in the use of the rodent guillotine (Thomas Scientific), Q2 221 222 serum and ovaries were collected. Right ovaries were stored 223 at 80 C until assayed. Left ovaries were fixed in 4% 224 paraformaldehyde-PBS (pH 7.4) and embedded in paraffin. 225 Trunk blood was collected and allowed to clot at 4 C. Serum 226 was obtained by centrifugation at 3500g for 15 min at 4 C 227 and stored at 20 C until assayed. 228 229 Follicular Count 230 231 Ten micrometer serial sections were obtained from the 232 complete ovary and stained with hematoxylin-eosin. Each 233 preantral and antral follicle was quantified throughout the 234 Materials and Methods
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whole ovary. To avoid repeated counts for a single follicle, only preantral and antral follicles showing a nucleolus in the oocyte were counted. Follicular classification was performed according to follicular cell layers, morphology and the presence or absence of an antral cavity into the follicle was designated a preantral follicles when two or more cuboidal follicular cell layers were seen surrounding the oocyte and no antral cavity was present. Follicles were classified as antral when two or more cuboidal follicular cell layers and an antral cavity were present. FSH and LH Serum Concentrations Serum LH and FSH were determined by radioimmunoassay using hormone standards and specific antibodies provided by the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK, NIH, Bethesda, MD). FSH and LH were iodinated by the chloramine-T method followed by separation of protein bound and free 125I by Sephadex D-100. Samples of 100 ml per tube were assayed in duplicate. Results were expressed in terms of NIDDK-rat-FSH-RP2 and NIDDK-rat-LH-RP3 standards. Sensitivity was 0.035 and 0.02 ng assay/100ml, respectively. FSH intra- and interassay coefficients of variation were!9 and!14% respectively, and for LH!7 and!12%. RNA Isolation and mRNA Expression Total ovarian RNA was isolated by the guanidine isothiocyanate/phenol/chloroform extraction method using TRIzol reagent (Invitrogen, Carlsbad, CA). RNA concentration was spectrophotometrically quantified (l 5 260 nmol) and integrity determined by gel electrophoresis on 2% denaturing agarose in the presence of 2.2 M formaldehyde. Five mg of total RNA were reverse transcribed by using the SuperScript First Strand Synthesis System (Invitrogen) for RT-PCR. Cyclophilin gene was used as an endogenous control. Oligonucleotides used are shown in Table 1. Each PCR reaction was performed in a 25-ml volume that included 5 ml of previously synthesized cDNA, 0.1 mmol of each dNTP (Invitrogen), 0.1 mmol of each primer, and 2.5 units of Taq DNA polymerase (Roche Diagnostics). Negative controls without cDNA and with non-retrotranscribed RNA were included in all trials. Ten ml of PCR products was separated on 2% agarose gel and stained with ethidium bromide. The bands of the predicted sizes were confirmed by Southern blot analysis with
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specific rat cDNA probes isolated from adult rat ovaries and labeled with digoxigenin (DIG DNA labeling and detection Kit II, Roche Diagnostics) (20). Images were captured with a digital camera and quantified by densitometry using Vision Works Analysis Software (UVP, Bioimaging Systems). Expression levels were normalized to that of cyclophilin A (Cyc). Statistical Analysis Five and six litters were studied in each group (CC and RR: n 5 5; CR and RC n 5 6). One female per litter was analyzed. All data were expressed as mean SEM. Comparisons among groups were performed using one-way ANOVA followed by the Tukey post hoc test. Pearson correlation coefficient was obtained for the relationship between previously reported estradiol levels (8) and aromatase expression;p! 0.05 was considered significant.
Results Prepubertal Growth at 21 Days of Age At 21 days, body weight was lower in females whose mothers were restricted during lactation (RR and CR) and increased in RC compared to CC (CC 5 43 2; RR 5 28 0.4; CR 5 31 1; RC 5 51 2g; p !0.05). Ovary weight was higher in RC pups compared to RR (CC 5 11 0.9; RR 5 10 0.6; CR 5 11 0.8; RC 5 13 0.9 mg; p ! 0.05). When expressed as relative to body weight, RR ovaries were heavier compared to CC and RC (CC 5 0.026 0.002; RR 5 0.037 0.002; CR 5 0.03 0.001; RC 5 0.028 0.003%; p !0.05). Preantral and Antral Follicular Development at 21 Days of Age Numbers of preantral and antral follicles were higher in RR ovaries compared to CR and RC (Figures 1A and 1C). No differences were found between the number of preantral and antral follicles of the same group. Interestingly, the adjustment of the number of preantral and antral follicles to the ovarian weight showed that RR females possessed a significantly higher number of follicles compared to groups restricted only during pregnancy or lactation. These two
Table 1. Oligonucleotide sequences
StAR P450scc P450 aromatase LH-R FSH-R Cyclophilin A
Forward primer
Reverse primer
Product size (bp)
50 -CCA GCA AGG AGA GGA AGC TAT-30 50 -ATG CTG GCA AAA GGT CTT T-30 50 -CCT GGC AAG CAC TCC TTA TC-30 50 -GGA TGC TTT CCA AGG GAT GA-30 50 -GAG TCA TCC CGA AAG GAT CA-30 50 -CCC CAC CGT GTT CTT CGA CAT-30
50 -AGC ACA CAG GTG GAA CCT CT-30 50 -CCT GTA AAT GGG GCC ATA-30 50 -TAC CGC AGG ATA TCG TTA AT-30 50 -CCT CAA AGA TGG CGG AAT AA-30 50 -TAA AAT GAC TGG CCC AGA GG-30 50 -GCT GGT CTT GCC ATT CCT GGA-30
370 310 430 470 494 451
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Figure 1. Follicular development in the 21-day-old ovary. Female pups of 21 days from mothers fed a control (C, 20% casein) or restricted (R, 10% casein) isocaloric diet during pregnancy (first letter) and lactation (second letter). (A) Preantral and antral follicular numbers. (B) Preantral and antral number of follicles adjusted to the ovarian weight. (C) Representative micrograph per group at 40X magnification. Mean SEM; n 5 5e6 litters. p !0.05 data not sharing at least one letter are statistically different from each other.
groups (CR and RC) presented lower number of follicles compared to CC and RR (Figures 1B and 1C). FSH and LH Serum Concentrations Serum LH concentration was lower (Figure 2A) and FSH was higher in CR group compared to CC pups (Figure 2B). Gonadotropin Receptor mRNA Expression in the Ovary LH-R relative mRNA expression was reduced in RR ovaries compared to CC, CR and RC groups (Figure 2C). FSH-R expression did not show any difference among groups (Figure 2D). StAR, P450scc, P450 Aromatase mRNA Expression in the Ovary Relative StAR mRNA expression was higher in the ovaries of pups from mothers who were protein restricted during either pregnancy (RC) or lactation (CR) in comparison to CC and RR (Figure 3A). P450scc expression was lower in RR and increased in RC compared to CC and CR (Figure 3B). In contrast, P450 aromatase expression was lower in RR and CR groups compared to CC and RC (Figure 3C). As reported elsewhere in this model (8), serum
estradiol is reduced in RR and CR female pups at 21 days of age. A positive correlation between P450 aromatase expression and previously observed estradiol serum levels was observed (n 5 4 pups form different litters, r2 5 0.667, p !0.005).
Discussion Maternal nutrition during early offspring development affects reproductive function in male (2,4,5,21,22) and female rat offspring (8,22,23). In this report we show that maternal protein restriction during pregnancy and/or lactation alters daughters’ preantral and antral follicular numbers as well as ovarian function in the prepubertal stage of development. Female offspring from mothers who were protein restricted during either pregnancy, lactation or both periods exhibit altered estrous cycles and premature reproductive aging (8). Pubertal development has also been shown to be modified by maternal nutrition because onset of puberty is delayed in female rat pups exposed to maternal nutrient restriction during lactation (1,8). Our previous data show that the delay in the onset of puberty was 4 and 2 days in RR and CR, respectively, in comparison with CC (8).
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Figure 2. LH and FSH serum levels and LH-R and FSH-R mRNA expression in the 21-day-old ovary. Female pups of 21 days fro m mothers fed a control (C, 20% casein) or restricted (R, 10% casein) isocaloric diet during pregnancy (first letter) and lactation (second letter). (A) LH. (B) FSH serum levels (n 5 5e6 litters). (C) LH-R and D. FSH-R ovary relative expression (n 5 4 pups from different litters). Mean SEM; p !0.01 data not sharing at least one letter are statistically different from each other.
Lower prepubertal body, ovarian and uterine weights are likely early signs of delayed sexual development (8), but other changes we describe here are also probably implicated directly in impaired ovarian function and could be key causes of ovarian dysfunction. In rats, ovarian follicular structures are formed in the early days of postnatal life. In the first 3 weeks after birth, the first wave of follicles develop from primordial to antral (11,12) and, by postnatal day 18 when antral follicles are present in the ovary, an important wave of follicular apoptosis is observed as a normal cue (12,14). We assessed the preantral and antral follicular numbers in ovaries of prepubertal 21-day-old rats. Greater numbers of these types of follicles were found in pups from mothers restricted during
both pregnancy and lactation (RR) compared with those whose mothers were restricted for only one period (RC or CR), suggesting that maternal nutrition affects follicular growth in a different manner according to the stage during which the mother is restricted. The environmental challenge represented by the change in maternal diet could increase ovarian loss of follicles during lactation that is important in programming sexual development and reproductive capacity of the female offspring. It has been suggested that when the environment during postnatal life does not match the environment experienced in utero, individuals develop a maladapted phenotype (24) as occurs in relation to longevity and fertility in the female rat (8). In contrast, maintaining the protein restricted diet during both
Figure 3. mRNA expression of StAR, P450 scc and P450 aromatase in the 21-day-old ovary. Female pups of 21 days from mothers fed a control (C, 20% casein) or restricted (R, 10% casein) diet during pregnancy (first letter) and lactation (second letter). (A) StAR. (B) P450 scc. (C) P450 aromatase. Mean SEM; n 5 4 pups from different litters. p !0.001 for data not sharing at least one letter.
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pregnancy and lactation significantly delays sexual development (8), but here we show that it does not diminish prepubertal preantral and antral follicular counts, which could be a sign of delayed ovarian developmental apoptosis and ovarian function. Follicular development and onset of puberty are dependent on gonadotropins (17,25). Both FSH and LH levels as well as their receptors play key roles during the prepubertal stage of ovarian development prior to initiation of ovulation and cyclic follicular growth (17). At 21 days of age, RR females have LH and FSH serum levels similar to the control group (CC) but lower LH-R expression. In contrast, at this prepubertal age, CR females exhibited decreased LH and increased FSH levels but no different expression of their receptors. It is likely that these data explain, at least in part, why RR and CR groups of females were unable, in comparison with the CC group, to achieve puberty on time and that sexual maturation was delayed due to inadequate gonadotropin stimulation to the ovary during the prepubertal stage. The data reported here suggest that there are different mechanisms delaying ovarian maturation and consequently puberty in the presence of the different nutritional exposures. In RR females the cause of this delay seems to be at the ovarian level resulting from lower LH-R expression, whereas in CR the cause may be lower pituitary LH secretion. Maternal nutrition during lactation seems to be crucial for sexual and ovarian development as well as its prepubertal function. Maternal protein restriction during this stage of development impairs the first wave of follicular growth and prepubertal steroidogenesis that could explain the sexual maturation delay observed in these female pups (8). At the ovarian level, changes in the steroidogenic response to gonadotropins are mediated by LH and FSH receptor contents in the ovary (26). During the early weeks of postnatal development, the rat ovary increases its contents of FSH-R and this is induced by FSH and estrogen levels (27). FSH interaction with its receptor exerts the transcriptional control of the P450 aromatase (26,28). At 21 days, the main effect observed in RR and CR female offspring is on estrogen synthesis, but progesterone levels have also shown to be lower (8). In this study we observed changes in expression of some genes involved in the steroidogenic pathway. Regardless of the period of maternal restriction, expression of StAR was higher in CR and RC restricted groups. P450 scc, the initial steroidogenic step, was lower in pups from RR mothers and higher in RC, which is consistent with the timing of sexual maturation observed in each group. A positive correlation between ovarian P450 aromatase expression and serum estradiol at 21 days of age was found. Environmental challenges during pregnancy, including malnutrition and over exposure to steroids, have been associated with several alterations in the female reproductive axis, particularly ovarian function (1,3,8,29e36). During
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lactation, environmental factors also play key roles in ovarian development. Maternal nutrition can modify sexual development and ovarian dynamics (37,38). In our previous report, fertility did not show affection at the age of 150 days in any of the groups, whereas at 1 year a dramatic drop in fertility rate was observed in CR and RC females (8). Coincidentally, we are reporting here that CR and RC females exhibit the lower numbers of both preantral and antral follicular population at postnatal day 21. It is possible that these groups in which fetal and neonatal environment do not match may, in fact, possess a lower endowment of total follicles. It seems that these females exert their maximal reproductive capacities at a younger age, although it could diminish their performance earlier in life compared to control females. The first 3 weeks of postnatal life are crucial in the development of ovarian follicles in the rat (11,15). It is probable that the follicular pools established at birth are diminished by changing the maternal diet 0and that lactation corresponds to the critical window of development for ovarian follicles. In this sense, fertility rate could be set during this stage of development in the female rat, but further studies are necessary to identify the mechanism that underlies the programming of follicular development, ovarian function and fertility rate. In conclusion, in the rat, the first 3 weeks of postnatal life are crucial in the development of ovarian follicles. Our data suggest that maintaining the restricted diet during both pregnancy and lactation delays the onset of puberty by mechanisms affecting mainly the ovarian function. In contrast, the environmental challenge represented by the change in maternal diet during lactation not only impacts ovarian function but also affects other levels of the hypothalamicpituitary-ovarian axis. Both RR and CR nutritional schemes lead to the same observed finding of delayed sexual maturation by different mechanisms. References 1. Leonhardt M, Lesage J, Croix D, et al. Effects of perinatal maternal food restriction on pituitary-gonadal axis and plasma leptin level in rat pup at birth and weaning and on timing of puberty. Biol Reprod 2003;68:390e400. 2. Zambrano E, Rodriguez-Gonzalez GL, Guzman C, et al. A maternal low protein diet during pregnancy and lactation in the rat impairs male reproductive development. J Physiol 2005;563:275e284. 3. Gunn R. Effects of nutrition in utero and in early life on the subsequent lietime reproductive performance of Scottish Blackface ewes in two management systems. Anim Sci 1995;60:223e230. 4. Connor KL, Vickers MH, Beltrand J, et al. Nature, nurture or nutrition? Impact of maternal nutrition on maternal care, offspring development and reproductive function. J Physiol 2012;590:2167e2180. 5. Bernal AB, Vickers MH, Hampton MB, et al. Maternal undernutrition significantly impacts ovarian follicle number and increases ovarian oxidative stress in adult rat offspring. PLoS One 2010;5:e15558. 6. Rhind SM. Effects of maternal nutrition on fetal and neonatal reproductive development and function. Anim Reprod Sci 2004;82e83:169e181. 7. Rhind SM, Rae MT, Brooks AN. Effects of nutrition and environmental factors on the fetal programming of the reproductive axis. Reproduction 2001;122:205e214.
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8. Guzman C, Cabrera R, Cardenas M, et al. Protein restriction during fetal and neonatal development in the rat alters reproductive function and accelerates reproductive ageing in female progeny. J Physiol 2006; 572:97e108. 9. Frisch RE, Hegsted DM, Yoshinaga K. Body weight and food intake at early estrus of rats on a high-fat diet. Proc Natl Acad Sci USA 1975; 72:4172e4176. 10. Nunez-de la Mora A, Chatterton RT, Choudhury OA, et al. Childhood conditions influence adult progesterone levels. PLoS Med 2007;4: e167. 11. McGee EA, Hsueh AJ. Initial and cyclic recruitment of ovarian follicles. Endocr Rev 2000;21:200e214. 12. McGee EA, Hsu SY, Kaipia A, et al. Cell death and survival during ovarian follicle development. Mol Cell Endocrinol 1998;140:15e18. 13. Merchant-Larios H, Chimal-Monroy J. The ontogeny of primordial follicles in the mouse ovary. Prog Clin Biol Res 1989;296:55e63. 14. Hsueh AJ, McGee EA, Hayashi M, et al. Hormonal regulation of early follicle development in the rat ovary. Mol Cell Endocrinol 2000;163: 95e100. 15. McGee EA, Perlas E, LaPolt PS, et al. Follicle-stimulating hormone enhances the development of preantral follicles in juvenile rats. Biol Reprod 1997;57:990e998. 16. Urbanski HF, Ojeda SR. In vitro simulation of prepubertal changes in pulsatile luteinizing hormone release enhances progesterone and 17 beta-estradiol secretion from immature rat ovaries. Endocrinology 1985;117:638e643. 17. Smith SS, Ojeda SR. Neonatal release of gonadotropins is essential for development of ovarian follicle-stimulating hormone receptors. Biol Reprod 1986;34:219e227. 18. Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev 1996;17:121e155. 19. Hirshfield AN. Development of follicles in the mammalian ovary. Int Rev Cytol 1991;124:43e101. 20. Mendez I, Alcocer-Varela J, Parra A, et al. Neuroendocrine dopaminergic regulation of prolactin release in systemic lupus erythematosus: a possible role of lymphocyte-derived prolactin. Lupus 2004;13: 45e53. 21. Vigueras-Villasenor RM, Rojas-Castaneda JC, Chavez-Saldana M, et al. Alterations in the spermatic function generated by obesity in rats. Acta Histochem 2011;113:214e220. 22. Zambrano E, Guzman C, Rodriguez-Gonzalez GL, et al. Fetal programming of sexual development and reproductive function. Mol Cell Endocrinol 2014;382:538e549. 23. Sloboda DM, Howie GJ, Pleasants A, et al. Pre- and postnatal nutritional histories influence reproductive maturation and ovarian function in the rat. PLoS One 2009;4:e6744. 24. Ferreira RV, Gombar FM, da Silva Faria T, et al. Metabolic programming of ovarian angiogenesis and folliculogenesis by maternal malnutrition during lactation. Fertil Steril 2010;93:2572e2580.
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25. Ojeda SR, Urbanski HF, Katz KH, et al. Activation of estradiol-positive feedback at puberty: estradiol sensitizes the LHRH-releasing system at two different biochemical steps. Neuroendocrinology 1986;43: 259e265. 26. Richards JS. Hormonal control of gene expression in the ovary. Endocr Rev 1994;15:725e751. 27. Channing CP, Schaerf FW, Anderson LD, et al. Ovarian follicular and luteal physiology. Int Rev Physiol 1980;22:117e201. 28. Hickey GJ, Chen SA, Besman MJ, et al. Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: relationship to estradiol biosynthesis. Endocrinology 1988;122: 1426e1436. 29. Harvey PW, Chevins PF. Deleterious effects of adrenocorticotrophic hormone administration during late pregnancy upon offspring somatic, neurological, and sexual development in mice. Teratology 1987;35: 229e238. 30. Iwasa T, Matsuzaki T, Murakami M, et al. Delayed puberty in prenatally glucocorticoid administered female rats occurs independently of the hypothalamic Kiss1-Kiss1r-GnRH system. Int J Dev Neurosci 2011;29:183e188. 31. Kosut SS, Wood RI, Herbosa-Encarnacion C, et al. Prenatal androgens time neuroendocrine puberty in the sheep: effect of testosterone dose. Endocrinology 1997;138:1072e1077. 32. Luense LJ, Veiga-Lopez A, Padmanabhan V, et al. Developmental programming: gestational testosterone treatment alters fetal ovarian gene expression. Endocrinology 2011;152:4974e4983. 33. Manikkam M, Steckler TL, Welch KB, et al. Fetal programming: prenatal testosterone treatment leads to follicular persistence/luteal defects; partial restoration of ovarian function by cyclic progesterone treatment. Endocrinology 2006;147:1997e2007. 34. Smith JT, Waddell BJ. Increased fetal glucocorticoid exposure delays puberty onset in postnatal life. Endocrinology 2000;141:2422e2428. 35. Steckler T, Wang J, Bartol FF, et al. Fetal programming: prenatal testosterone treatment causes intrauterine growth retardation, reduces ovarian reserve and increases ovarian follicular recruitment. Endocrinology 2005;146:3185e3193. 36. Veiga-Lopez A, Steckler TL, Abbott DH, et al. Developmental programming: impact of excess prenatal testosterone on intrauterine fetal endocrine milieu and growth in sheep. Biol Reprod 2011;84:87e96. 37. Faria Tda S, Brasil Fde B, Sampaio FJ, et al. Maternal malnutrition during lactation alters the folliculogenesis and gonadotropins and estrogen isoforms ovarian receptors in the offspring at puberty. J Endocrinol 2008;198:625e634. 38. da Silveira Cavalcante F, Gombar FM, Ferreira RV, et al. Maternal protein-energy and energy-restricted diets during lactation possibly could program folliculogenesis and the ovarian expression of leptin and its different isoform receptors in rats. Fertil Steril 2009;92: 1755e1757.
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ORIGINAL ARTICLE
Maternal Protein Restriction During Pregnancy and/or Lactation Negatively Affects Follicular Ovarian Development and Steroidogenesis in the Prepubertal Rat Offspring Q3
Carolina Guzman,a Rocıo Garcıa-Becerra,b Marco Antonio Aguilar-Medina,b Isabel Mendez,c Horacio Merchant-Larios,d and Elena Zambranob
a
HIPAM, Unidad de Medicina Experimental, Hospital General de Mexico, Facultad de Medicina, Universidad Nacional Autonoma de Mexico (UNAM), Mexico, D.F., Mexico b Departamento de Biologıa de la Reproduccion, Instituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiran, Mexico, D.F., Mexico c Instituto de Neurobiologıa, UNAM, Campus UNAM-Juriquilla, Mexico d Departamento de Biologıa Celular y Fisiologıa, Instituto de Investigaciones Biomedicas, UNAM, Mexico, D.F., Mexico Received for publication November 12, 2013; accepted March 14, 2014 (ARCMED-D-13-00640).
Background and Aims. Maternal protein restriction during rat pregnancy and lactation is associated with alterations in reproductive function of female offspring including delayed onset of puberty, decreased fertility and premature reproductive aging. These alterations may be related to ovarian prepubertal development, distribution of follicle populations and their steroidogenic capacities. We undertook this study to evaluate the ovarian function of prepubertal female offspring exposed to maternal protein restriction during pregnancy and/or lactation. Methods. Adult female Wistar rats were fed a control (C-20% casein diet) or restricted isocaloric diet (R-10% casein) during pregnancy—first letter—and lactation—second letter, to form four groups, CC, RR, CR, RC. Ovaries were collected from 21-day-old female offspring. Preantral and antral follicles were quantified and mRNA expression of key genes involved in follicular development and steroidogenesis (gonadotropin receptors, StAR, P450scc and P450 aromatase) was evaluated. Serum gonadotropin levels were measured. Results. Significantly decreased numbers of preantral and antral follicles were observed in CR and RC ovaries compared with CC. LH levels were lower and FSH higher in CR pups. mRNA expression of LH receptor (LH-R) was decreased in RR in comparison with the other groups. CR and RC expressed higher StAR, RC increased and RR decreased P450scc, whereas RR and CR decreased aromatase expression in comparison with CC. Conclusions. Maternal protein restriction influences prepubertal ovarian follicular number and steroidogenic function in the rat offspring, although RR and CR nutritional schemes have similar outcomes, the mechanisms affecting ovarian function are at different levels of the hypothalamic-pituitary-ovarian axis. Ó 2014 IMSS. Published by Elsevier Inc. Key Words: Maternal protein restriction, Ovary, Prepubertal development, Developmental programming.
Introduction
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Addressed reprint requests to: Dr. Elena Zambrano, Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiran, Vasco de Quiroga 15, Tlalpan 14000, Mexico, D.F. Mexico; E-mail: [email protected]
Programming of reproductive function has been demonstrated in response to exposure to a suboptimal environment during fetal and postnatal development (1e7). Maternal
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protein restriction during rat pregnancy alters key components of maternal steroid function increasing fetal exposure to estradiol, testosterone, progesterone and corticosterone (2,8). In the female offspring, onset of puberty and estrous cycles are delayed (1,8), fertility rates decreased (3,8), and reproductive lifespan shortened (8). Nutritional challenges during early life have been associated with altered pubertal development. Maternal protein restriction during lactation in the rat delays vaginal opening and first estrous, independently of maternal nutrition during pregnancy (8). In contrast, females fed high fat from weaning have advanced onset of puberty (4,9). In humans, migration has allowed researchers to identify the developmental effects of environmental changes during early life. For example, Bangladeshi girls raised in the UK have higher salivary progesterone levels if they migrate earlier than menarche. When migration occurs at a later age, they show lower progesterone salivary levels and later pubertal maturation (10). Sexual maturation and fertility depend on an adequate ovarian function, which is based on the availability of a pool of follicles for recruitment and ovulation (11) as well as an appropriate hormonal environment. In rats, follicular development begins in neonatal life when follicular structures are established in numbers that will progressively decrease through the reproductive lifespan (11,12). Ovarian follicles contain an oocyte surrounded by follicular cells. The numbers of follicular cell layers depends on the stage of follicular development (11,13,14). Follicles are classified according to the layers of granulosa cells as well as presence or absence of the antral cavity (15). In the rat, well-developed antral follicles are present by post natal day 15e17 (12). Ovarian steroidogenesis starts before puberty. Prepubertal estradiol levels regulate the establishment of pulsatile LH secretion (16). Both gonadotropins, LH and FSH, regulate expression of their receptors and the enzymes involved in sex steroid synthesis (17). Antral follicles express LH and FSH receptors and are capable of aromatization (14). Most of the prepubertal antral follicles undergo atresia, only a few reach the preovulatory stage under gonadotropin stimulation (11,14,18,19). FSH prevents programmed demise in early antral follicles (14). We hypothesized that assessment of prepubertal ovarian development and function could provide an early indicator of programming of sexual development due by maternal protein restriction during pregnancy and/or lactation. We assessed the number of the ovarian preantral and antral follicles in ovaries of 21-day-old offspring exposed to maternal protein restriction during pregnancy and/or lactation. Expression of ovarian proteins involved in follicular development and steroidogenesis (LH receptor (LH-R), FSH receptor (FSH-R), steroidogenic acute regulator (StAR), P450 side chain cleavage (P450scc) and P450 aromatase), as well as LH and FSH serum levels were measured.
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180 181 Care of Animals 182 183 Details of protein restriction and generation of pups have 184 been published previously (2). Briefly, 3-month-old virgin 185 female albino Wistar rats (240e260 g) were obtained from 186 the Instituto Nacional de Ciencias Medicas y Nutrici on Sal187 vador Zubiran (Mexico City, Mexico) and maintained under 188 controlled lighting and temperature (lights on 07:00 h to 189 19:00 h at 22e23 C). All procedures were approved by 190 the Institutional Animal Experimentation Ethics Committee 191 of the Instituto Nacional de Ciencias Medicas y Nutrici on 192 Salvador Zubiran, Mexico City and are in accordance with 193 Mexican and international laws for laboratory animal care. 194 Rats were mated overnight with proven male breeders 195 and the day spermatozoa were present in the vaginal smear 196 was designated day of conception, day 0. Only rats 197 becoming pregnant within 5 days of mating were studied. 198 Pregnant rats were transferred to individual cages and allo199 cated at random to one of two groups fed either 20% casein 200 (control dieteC) or 10% casein isocaloric diet (restricted 201 dieteR) (2). Food was provided as large flat biscuits re202 tained behind a grill through which rats nibbled the food. 203 Food and water were available ad libitum. 204 Vaginal delivery occurred spontaneously in the early 205 daylight hours between 09:00 and 12:00 h on post206 conceptual day 22. Day of delivery was considered as day 207 0 of postnatal life. Timing of delivery, litter size and pup 208 weight were recorded at birth. Anogenital distance was as209 sessed to determine pup sex using our previous criteria (8). 210 Sex was judged according to whether anogenital distance 211 was lower (female) or larger (male) than 2.5 mm. To ensure 212 homogeneity of study subjects, litters over 14 pups were not 213 included. Litters of 12e14 pups were adjusted to 12 pups for 214 each dam, maintaining as close to a 1:1 sex ratio as possible. 215 Four groups were established CC, RR, CR and RC. First let216 ter refers to maternal diet in pregnancy and second diet in lac217 tationeC control, R restricted. We report data on female 218 offspring only. 219 At 21 days of postnatal life, one female pup per litter was 220 rapidly euthanized by decapitation by experienced personnel trained in the use of the rodent guillotine (Thomas Scientific), Q2 221 222 serum and ovaries were collected. Right ovaries were stored 223 at 80 C until assayed. Left ovaries were fixed in 4% 224 paraformaldehyde-PBS (pH 7.4) and embedded in paraffin. 225 Trunk blood was collected and allowed to clot at 4 C. Serum 226 was obtained by centrifugation at 3500g for 15 min at 4 C 227 and stored at 20 C until assayed. 228 229 Follicular Count 230 231 Ten micrometer serial sections were obtained from the 232 complete ovary and stained with hematoxylin-eosin. Each 233 preantral and antral follicle was quantified throughout the 234 Materials and Methods
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whole ovary. To avoid repeated counts for a single follicle, only preantral and antral follicles showing a nucleolus in the oocyte were counted. Follicular classification was performed according to follicular cell layers, morphology and the presence or absence of an antral cavity into the follicle was designated a preantral follicles when two or more cuboidal follicular cell layers were seen surrounding the oocyte and no antral cavity was present. Follicles were classified as antral when two or more cuboidal follicular cell layers and an antral cavity were present. FSH and LH Serum Concentrations Serum LH and FSH were determined by radioimmunoassay using hormone standards and specific antibodies provided by the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK, NIH, Bethesda, MD). FSH and LH were iodinated by the chloramine-T method followed by separation of protein bound and free 125I by Sephadex D-100. Samples of 100 ml per tube were assayed in duplicate. Results were expressed in terms of NIDDK-rat-FSH-RP2 and NIDDK-rat-LH-RP3 standards. Sensitivity was 0.035 and 0.02 ng assay/100ml, respectively. FSH intra- and interassay coefficients of variation were!9 and!14% respectively, and for LH!7 and!12%. RNA Isolation and mRNA Expression Total ovarian RNA was isolated by the guanidine isothiocyanate/phenol/chloroform extraction method using TRIzol reagent (Invitrogen, Carlsbad, CA). RNA concentration was spectrophotometrically quantified (l 5 260 nmol) and integrity determined by gel electrophoresis on 2% denaturing agarose in the presence of 2.2 M formaldehyde. Five mg of total RNA were reverse transcribed by using the SuperScript First Strand Synthesis System (Invitrogen) for RT-PCR. Cyclophilin gene was used as an endogenous control. Oligonucleotides used are shown in Table 1. Each PCR reaction was performed in a 25-ml volume that included 5 ml of previously synthesized cDNA, 0.1 mmol of each dNTP (Invitrogen), 0.1 mmol of each primer, and 2.5 units of Taq DNA polymerase (Roche Diagnostics). Negative controls without cDNA and with non-retrotranscribed RNA were included in all trials. Ten ml of PCR products was separated on 2% agarose gel and stained with ethidium bromide. The bands of the predicted sizes were confirmed by Southern blot analysis with
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specific rat cDNA probes isolated from adult rat ovaries and labeled with digoxigenin (DIG DNA labeling and detection Kit II, Roche Diagnostics) (20). Images were captured with a digital camera and quantified by densitometry using Vision Works Analysis Software (UVP, Bioimaging Systems). Expression levels were normalized to that of cyclophilin A (Cyc). Statistical Analysis Five and six litters were studied in each group (CC and RR: n 5 5; CR and RC n 5 6). One female per litter was analyzed. All data were expressed as mean SEM. Comparisons among groups were performed using one-way ANOVA followed by the Tukey post hoc test. Pearson correlation coefficient was obtained for the relationship between previously reported estradiol levels (8) and aromatase expression;p! 0.05 was considered significant.
Results Prepubertal Growth at 21 Days of Age At 21 days, body weight was lower in females whose mothers were restricted during lactation (RR and CR) and increased in RC compared to CC (CC 5 43 2; RR 5 28 0.4; CR 5 31 1; RC 5 51 2g; p !0.05). Ovary weight was higher in RC pups compared to RR (CC 5 11 0.9; RR 5 10 0.6; CR 5 11 0.8; RC 5 13 0.9 mg; p ! 0.05). When expressed as relative to body weight, RR ovaries were heavier compared to CC and RC (CC 5 0.026 0.002; RR 5 0.037 0.002; CR 5 0.03 0.001; RC 5 0.028 0.003%; p !0.05). Preantral and Antral Follicular Development at 21 Days of Age Numbers of preantral and antral follicles were higher in RR ovaries compared to CR and RC (Figures 1A and 1C). No differences were found between the number of preantral and antral follicles of the same group. Interestingly, the adjustment of the number of preantral and antral follicles to the ovarian weight showed that RR females possessed a significantly higher number of follicles compared to groups restricted only during pregnancy or lactation. These two
Table 1. Oligonucleotide sequences
StAR P450scc P450 aromatase LH-R FSH-R Cyclophilin A
Forward primer
Reverse primer
Product size (bp)
50 -CCA GCA AGG AGA GGA AGC TAT-30 50 -ATG CTG GCA AAA GGT CTT T-30 50 -CCT GGC AAG CAC TCC TTA TC-30 50 -GGA TGC TTT CCA AGG GAT GA-30 50 -GAG TCA TCC CGA AAG GAT CA-30 50 -CCC CAC CGT GTT CTT CGA CAT-30
50 -AGC ACA CAG GTG GAA CCT CT-30 50 -CCT GTA AAT GGG GCC ATA-30 50 -TAC CGC AGG ATA TCG TTA AT-30 50 -CCT CAA AGA TGG CGG AAT AA-30 50 -TAA AAT GAC TGG CCC AGA GG-30 50 -GCT GGT CTT GCC ATT CCT GGA-30
370 310 430 470 494 451
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Figure 1. Follicular development in the 21-day-old ovary. Female pups of 21 days from mothers fed a control (C, 20% casein) or restricted (R, 10% casein) isocaloric diet during pregnancy (first letter) and lactation (second letter). (A) Preantral and antral follicular numbers. (B) Preantral and antral number of follicles adjusted to the ovarian weight. (C) Representative micrograph per group at 40X magnification. Mean SEM; n 5 5e6 litters. p !0.05 data not sharing at least one letter are statistically different from each other.
groups (CR and RC) presented lower number of follicles compared to CC and RR (Figures 1B and 1C). FSH and LH Serum Concentrations Serum LH concentration was lower (Figure 2A) and FSH was higher in CR group compared to CC pups (Figure 2B). Gonadotropin Receptor mRNA Expression in the Ovary LH-R relative mRNA expression was reduced in RR ovaries compared to CC, CR and RC groups (Figure 2C). FSH-R expression did not show any difference among groups (Figure 2D). StAR, P450scc, P450 Aromatase mRNA Expression in the Ovary Relative StAR mRNA expression was higher in the ovaries of pups from mothers who were protein restricted during either pregnancy (RC) or lactation (CR) in comparison to CC and RR (Figure 3A). P450scc expression was lower in RR and increased in RC compared to CC and CR (Figure 3B). In contrast, P450 aromatase expression was lower in RR and CR groups compared to CC and RC (Figure 3C). As reported elsewhere in this model (8), serum
estradiol is reduced in RR and CR female pups at 21 days of age. A positive correlation between P450 aromatase expression and previously observed estradiol serum levels was observed (n 5 4 pups form different litters, r2 5 0.667, p !0.005).
Discussion Maternal nutrition during early offspring development affects reproductive function in male (2,4,5,21,22) and female rat offspring (8,22,23). In this report we show that maternal protein restriction during pregnancy and/or lactation alters daughters’ preantral and antral follicular numbers as well as ovarian function in the prepubertal stage of development. Female offspring from mothers who were protein restricted during either pregnancy, lactation or both periods exhibit altered estrous cycles and premature reproductive aging (8). Pubertal development has also been shown to be modified by maternal nutrition because onset of puberty is delayed in female rat pups exposed to maternal nutrient restriction during lactation (1,8). Our previous data show that the delay in the onset of puberty was 4 and 2 days in RR and CR, respectively, in comparison with CC (8).
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Figure 2. LH and FSH serum levels and LH-R and FSH-R mRNA expression in the 21-day-old ovary. Female pups of 21 days fro m mothers fed a control (C, 20% casein) or restricted (R, 10% casein) isocaloric diet during pregnancy (first letter) and lactation (second letter). (A) LH. (B) FSH serum levels (n 5 5e6 litters). (C) LH-R and D. FSH-R ovary relative expression (n 5 4 pups from different litters). Mean SEM; p !0.01 data not sharing at least one letter are statistically different from each other.
Lower prepubertal body, ovarian and uterine weights are likely early signs of delayed sexual development (8), but other changes we describe here are also probably implicated directly in impaired ovarian function and could be key causes of ovarian dysfunction. In rats, ovarian follicular structures are formed in the early days of postnatal life. In the first 3 weeks after birth, the first wave of follicles develop from primordial to antral (11,12) and, by postnatal day 18 when antral follicles are present in the ovary, an important wave of follicular apoptosis is observed as a normal cue (12,14). We assessed the preantral and antral follicular numbers in ovaries of prepubertal 21-day-old rats. Greater numbers of these types of follicles were found in pups from mothers restricted during
both pregnancy and lactation (RR) compared with those whose mothers were restricted for only one period (RC or CR), suggesting that maternal nutrition affects follicular growth in a different manner according to the stage during which the mother is restricted. The environmental challenge represented by the change in maternal diet could increase ovarian loss of follicles during lactation that is important in programming sexual development and reproductive capacity of the female offspring. It has been suggested that when the environment during postnatal life does not match the environment experienced in utero, individuals develop a maladapted phenotype (24) as occurs in relation to longevity and fertility in the female rat (8). In contrast, maintaining the protein restricted diet during both
Figure 3. mRNA expression of StAR, P450 scc and P450 aromatase in the 21-day-old ovary. Female pups of 21 days from mothers fed a control (C, 20% casein) or restricted (R, 10% casein) diet during pregnancy (first letter) and lactation (second letter). (A) StAR. (B) P450 scc. (C) P450 aromatase. Mean SEM; n 5 4 pups from different litters. p !0.001 for data not sharing at least one letter.
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pregnancy and lactation significantly delays sexual development (8), but here we show that it does not diminish prepubertal preantral and antral follicular counts, which could be a sign of delayed ovarian developmental apoptosis and ovarian function. Follicular development and onset of puberty are dependent on gonadotropins (17,25). Both FSH and LH levels as well as their receptors play key roles during the prepubertal stage of ovarian development prior to initiation of ovulation and cyclic follicular growth (17). At 21 days of age, RR females have LH and FSH serum levels similar to the control group (CC) but lower LH-R expression. In contrast, at this prepubertal age, CR females exhibited decreased LH and increased FSH levels but no different expression of their receptors. It is likely that these data explain, at least in part, why RR and CR groups of females were unable, in comparison with the CC group, to achieve puberty on time and that sexual maturation was delayed due to inadequate gonadotropin stimulation to the ovary during the prepubertal stage. The data reported here suggest that there are different mechanisms delaying ovarian maturation and consequently puberty in the presence of the different nutritional exposures. In RR females the cause of this delay seems to be at the ovarian level resulting from lower LH-R expression, whereas in CR the cause may be lower pituitary LH secretion. Maternal nutrition during lactation seems to be crucial for sexual and ovarian development as well as its prepubertal function. Maternal protein restriction during this stage of development impairs the first wave of follicular growth and prepubertal steroidogenesis that could explain the sexual maturation delay observed in these female pups (8). At the ovarian level, changes in the steroidogenic response to gonadotropins are mediated by LH and FSH receptor contents in the ovary (26). During the early weeks of postnatal development, the rat ovary increases its contents of FSH-R and this is induced by FSH and estrogen levels (27). FSH interaction with its receptor exerts the transcriptional control of the P450 aromatase (26,28). At 21 days, the main effect observed in RR and CR female offspring is on estrogen synthesis, but progesterone levels have also shown to be lower (8). In this study we observed changes in expression of some genes involved in the steroidogenic pathway. Regardless of the period of maternal restriction, expression of StAR was higher in CR and RC restricted groups. P450 scc, the initial steroidogenic step, was lower in pups from RR mothers and higher in RC, which is consistent with the timing of sexual maturation observed in each group. A positive correlation between ovarian P450 aromatase expression and serum estradiol at 21 days of age was found. Environmental challenges during pregnancy, including malnutrition and over exposure to steroids, have been associated with several alterations in the female reproductive axis, particularly ovarian function (1,3,8,29e36). During
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lactation, environmental factors also play key roles in ovarian development. Maternal nutrition can modify sexual development and ovarian dynamics (37,38). In our previous report, fertility did not show affection at the age of 150 days in any of the groups, whereas at 1 year a dramatic drop in fertility rate was observed in CR and RC females (8). Coincidentally, we are reporting here that CR and RC females exhibit the lower numbers of both preantral and antral follicular population at postnatal day 21. It is possible that these groups in which fetal and neonatal environment do not match may, in fact, possess a lower endowment of total follicles. It seems that these females exert their maximal reproductive capacities at a younger age, although it could diminish their performance earlier in life compared to control females. The first 3 weeks of postnatal life are crucial in the development of ovarian follicles in the rat (11,15). It is probable that the follicular pools established at birth are diminished by changing the maternal diet 0and that lactation corresponds to the critical window of development for ovarian follicles. In this sense, fertility rate could be set during this stage of development in the female rat, but further studies are necessary to identify the mechanism that underlies the programming of follicular development, ovarian function and fertility rate. In conclusion, in the rat, the first 3 weeks of postnatal life are crucial in the development of ovarian follicles. Our data suggest that maintaining the restricted diet during both pregnancy and lactation delays the onset of puberty by mechanisms affecting mainly the ovarian function. In contrast, the environmental challenge represented by the change in maternal diet during lactation not only impacts ovarian function but also affects other levels of the hypothalamicpituitary-ovarian axis. Both RR and CR nutritional schemes lead to the same observed finding of delayed sexual maturation by different mechanisms. References 1. Leonhardt M, Lesage J, Croix D, et al. Effects of perinatal maternal food restriction on pituitary-gonadal axis and plasma leptin level in rat pup at birth and weaning and on timing of puberty. Biol Reprod 2003;68:390e400. 2. Zambrano E, Rodriguez-Gonzalez GL, Guzman C, et al. A maternal low protein diet during pregnancy and lactation in the rat impairs male reproductive development. J Physiol 2005;563:275e284. 3. Gunn R. Effects of nutrition in utero and in early life on the subsequent lietime reproductive performance of Scottish Blackface ewes in two management systems. Anim Sci 1995;60:223e230. 4. Connor KL, Vickers MH, Beltrand J, et al. Nature, nurture or nutrition? Impact of maternal nutrition on maternal care, offspring development and reproductive function. J Physiol 2012;590:2167e2180. 5. Bernal AB, Vickers MH, Hampton MB, et al. Maternal undernutrition significantly impacts ovarian follicle number and increases ovarian oxidative stress in adult rat offspring. PLoS One 2010;5:e15558. 6. Rhind SM. Effects of maternal nutrition on fetal and neonatal reproductive development and function. Anim Reprod Sci 2004;82e83:169e181. 7. Rhind SM, Rae MT, Brooks AN. Effects of nutrition and environmental factors on the fetal programming of the reproductive axis. Reproduction 2001;122:205e214.
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