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¹ Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 14000, México, DF México
² Department of Biology, Faculty of Chemistry, Universidad Nacional Autónoma de México, CP 04510, México, DF México
³ Center for Pregnancy and Newborn Research, University of Texas Health Sciences Center San Antonio, TX 78229, USA

	Abstract

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Nutrient restriction during pregnancy and lactation impairs growth and development. Recent studies demonstrate long-term programming of function of specific organ systems resulting from suboptimal environments during fetal life and development up to weaning. We determined effects of maternal protein restriction (50% control protein intake) during fetal development and/or lactation in rats on the reproductive system of male progeny. Rats were fed either a control 20% casein diet (C) or a restricted diet (R) of 10% casein during pregnancy. After delivery mothers received either C or R diet until weaning to provide four groups: CC, RR, CR and RC. We report findings in male offspring only. Maternal protein restriction increased maternal serum corticosterone, oestradiol and testosterone (T) concentrations at 19 days gestation. Pup birth weight was unchanged but ano-genital distance was increased by maternal protein restriction (P < 0.05). Testicular descent was delayed 4.4 days in RR, 2.1 days in CR and 2.2 days in RC and was not related to body weight. Body weight and testis weight were reduced in RR and CR groups at all ages with the exception of CR testis weight at 270 days postnatal age (PN). At 70 days PN luteinizing hormone and T concentrations were reduced in RR, CR and RC. mRNA for P450 side chain cleavage (P450scc) was reduced in RR and CR at 21 days PN but was unchanged at 70 days PN. Fertility rate was reduced at 270 days PN in RC and sperm count in RR and RC. We conclude that maternal protein delays sexual maturation in male rats and that some effects only emerge in later life.

(Received 4 November 2004; accepted after revision 14 December 2004; first published online 20 December 2004)
Corresponding author E. Zambrano: Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 14000, México, DF México. Email: zamgon{at}laneta.apc.org

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Nutrient restriction during pregnancy and lactation impairs overall fetal growth and development. Investigations in several species of rodent and sheep have demonstrated long-term developmental programming of the function of specific organ systems as a result of fetal and neonatal exposure to a suboptimal environment (Hoet & Hanson, 1999). A variety of nutrient restriction protocols have been evaluated. In the several protein restriction models studied birth weight is usually lowered (Galler & Tonkiss, 1991; Langley & Jackson, 1994; Holemans et al. 1999; Vickers et al. 2001a, b; Woods et al. 2001). However, some studies report an increase in birth weight (Langley-Evans et al. 1996) or no effect (Langley & Jackson, 1994) resulting from maternal protein restriction. Birth weight is, however, a crude indicator of the degree of developmental compromise since, when challenged by adverse circumstances, developing mammals will recruit adaptive mechanisms to protect growth, especially of vital organs, during development (Cohn et al. 1974).

The majority of studies of developmental programming by maternal dietary restriction have investigated the cardiovascular system. Adult offspring of dams fed a 50% reduced protein diet showed elevated blood pressure (Sherman & Langley-Evans, 2000), but no elevation of blood pressure was found with a 33% reduction in protein (Langley et al. 1994). Protein restriction during development impairs vascular endothelial function in adult offspring (Brawley et al. 2003; Torrens et al. 2003).

Although a few reports in the sheep and rat clearly indicate impaired male sexual development, most of the data available on the effects of sub-optimal nutrition during development on reproductive function relate to the female (Gunn et al. 1995; Engelbregt et al. 2000; Alejandro et al. 2002; Rae et al. 2002; Leonhardt et al. 2003). We know of no data in rat models of protein restriction or any other challenge that addresses the issue of developmental programming of male fertility. In this study we evaluated effects of a low protein diet fed to rats during pregnancy and/or lactation on the development of the male reproductive system. To evaluate whether some of the consequences of exposure to a sub-optimal diet are not apparent in early postnatal life but take time to emerge, we followed the offspring for 270 days. We demonstrated delayed development of the male reproductive system and decreased fertility that emerged after 70 days of life.

	Methods

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Care and maintenance of animals

Seventy virgin female albino Wistar rats aged 10–12 weeks, weighing 220 ± 20 g (mean ± S.E.M.) were obtained from the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (Mexico City, Mexico). Rats were maintained under controlled lighting (lights on from 07.00 to 19.00 h) at 22–23°C. Prior to breeding, male and female rats were maintained on Purina Laboratory Chow 5001. All procedures were approved by the Animal Experimentation Ethics Committee of the Instituto Nacional de Ciencias Médicas y Nutrición, Salvador Zubirán, Mexico City.

Female rats were mated overnight with proven male breeders and the day on which spermatozoa were present in a vaginal smear was designated as day of conception – day 0. Only rats that were pregnant within 5 days of introduction of the male were retained in the study. Pregnant rats were transferred to individual metabolism cages and allocated at random to one of two groups to be fed either a 20% casein (control diet) or a 10% casein isocaloric (restricted) diet (Table 1). The diets were the same as those used extensively by other investigators except for the substitution of corn oil (Gloria, Corfuerte, S.A. de C.V, Mexico City) for soy (Reeves et al. 1993). In addition, we followed the American Institute of Nutrition's recommendation to supplement low protein diets with L-cystine (Sigma). Supplementation was proportional to the protein in the diet to ensure that the protein restriction was equivalent for all amino acids (Reeves et al. 1993). Thus the control diet contained 0.30% cystine and the protein-restricted diet 0.15% cystine (Table 1). Food and water were available ad libitum for all animals.

Experiment 1.  A group of 30 pregnant rats (13 controls and 17 protein-restricted mothers assigned at random) were studied for maternal and neonatal hormonal measurements. At 19 days gestation between 09.00 and 10.00 h, pregnant rats were bled from the tail vein while restrained in a small Plexiglass tube. Blood was collected into polyethylene tubes, allowed to clot at 4°C for 1 h, centrifuged at 3500 g for 15 min at 4°C and serum was stored at –30°C until assayed. Rats were then allowed to deliver vaginally. Pup weight was recorded at birth. Ano-genital distance was measured with calipers to enable determination of sex. To ensure homogeneity of study subjects, litters of over 14, or less than 12 pups, were excluded from the study. On day 1, litters of 12–14 pups were adjusted to 12 pups for each dam while maintaining as close to a 1 : 1 sex ratio as possible. Mothers maintained their pups for 2 days postnatally on the diets they ate during pregnancy. On day 2 of postnatal life pups were rapidly killed by decapitation between 09.00 and 10.00 h. Trunk blood was collected and processed as described above.

Experiment 2.  Twenty control pregnant rats and 20 protein-restricted pregnant rats underwent spontaneous vaginal delivery. Litter size and sex ratio was as in Experiment 1. After removal of five control litters (2 with less than 12 pups and 3 with more than 14) and six protein-restricted litters (3 with less than 12 pups and 3 with more than 14) from the study, pups from 23 of the remaining 29 mothers were used in the study. Four groups of pups were established: CC (n = 5) in which dams who received the control diet during pregnancy continued to be fed the control diet during lactation; RR (n = 5) in which dams who had received the restricted diet during pregnancy continued to receive the restricted diet during lactation; CR (n = 6) in which dams who received the control diet during pregnancy received the restricted diet during lactation; and RC (n = 7) in which dams who received the restricted diet during pregnancy were provided with the control diet during lactation. The other six litters were used in a separate study.

Postnatal maintenance

After weaning (postnatal day 21) all pups were fed with control diet ad libitum and maintained in groups of up to six until puberty. As the pups grew they were separated into groups of three to four. The pups were weighed daily and examined for completed testicular descent and preputial separation.

Between 09.00 and 10.00 h on days on which blood was sampled, rats were rapidly killed by decapitation by experienced personnel trained in the procedure using a rodent guillotine (Thomas Scientific, USA). Trunk blood was collected and processed as above. Testes were dissected and cleaned of fat and weighed.

Evaluation of reproductive function

Fertility of the male offspring.  Female rats with regular cycles were maintained on Purina Laboratory Chow 5001. To ensure that males and females received the same diet during the evaluation of fertility, females were placed on the control diet (Table 1) 1 week before being placed with the males. Females were mated individually for 5 days with adult male offspring at two different ages, 70–90 and 270 days old. Results were expressed as a percentage of female rats per group that became pregnant and reached 15 days gestation. Pregnant rats were then entered in another study.

Sperm count.  The epididymis was removed and cleaned of tissue and weighed. The tail of the epididymis was cut, weighed and dissociated with scissors in 1 ml of Ham's medium. Sperm count was performed in a Neubauer chamber in duplicate after dilution to 1 : 20 in Ham's medium.

RNA isolation.  Total RNA was isolated from the tissues as previously described (Chomczynski & Sacchi, 1987). Briefly, whole testes were homogenized in guanidinium isothiocyanate (Trizol) buffer. The phenol–chloroform-extracted RNA was quantified on a spectrometer at a wavelength of 260 nm. Twenty micrograms of total RNA were electrophoresed on a 1.2% agarose–formaldehyde gel under denaturing conditions and transferred to nylon membranes.

Northern blot analysis.  Effects of maternal protein restriction during pregnancy and/or lactation on P450scc mRNA expression in the offspring testes were analysed by Northern blot. Briefly, denatured RNA samples (20 µg) were subjected to 1.2% agarose gel electrophoresis in the presence of formaldehyde and transferred overnight by capillary blotting to a Zeta-Probe membrane (Bio-Rad). The RNA was fixed by UV cross-linking (Gene Cross-Linker; Bio-Rad) to the membrane. The blot was hybridized with a specific P450scc probe labelled with [³²P]-dCTP, at 65°C for 16 h.

The rat P450scc cDNA probe was generated as an RT-PCR product (310 bp) obtained using two sets of oligonucleotide primers: sense 5'-ATGCTGGCAAAAGGTCTTTGC-3' and antisense 5'-CCTGTAAATGGGGCCATACTT-3'. Numbering of the nucleotide base position for the P450scc gene was taken from GenBank profile accession number J05156 (rat P450scc mRNA).

The relative abundance of P450scc mRNA was determined by scanning the optical density of the corresponding signal on the autoradiographic film with the Bioimaging Systems software (LabWork version 4.5, UVP, Inc. Upland, CA, USA). 28S rRNA optical density was determined in the ethidium bromide-stained gel for each RNA sample, the ratio of P450scc mRNA signal to 28S rRNA signal was calculated, and group means were obtained.

Radioimmunoassays (RIAs)

Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations were determined by double-antibody RIA using hormone standards and specific anti-rat LH antibodies from the National Institute of Diabetes, Digestive and Kidney Diseases. Rat FSH and LH were iodinated by the chloramine-T method, following separation of protein-bound and free [¹²⁵I] by Sephadex G-100. Results were expressed in terms of NIDDK-rat-FSH-RP2 and NIDDK-rat-LH-RP3. The intra- and interassay coefficients of variations were < 5 and < 8% for FSH and < 7 and < 9% for LH.

Serum corticosterone, testosterone and oestradiol concentrations were determined by RIA using commercial rat kits, DPC Coat-a-count (TKRC1, TKTT1 and TKE21, respectively) from Diagnostic Products (Los Angeles, CA, USA). Intra- and interassay variabilities were < 6 and < 7%, < 5 and < 9% and < 6 and < 10% for corticosterone, testosterone and oestradiol, respectively.

Statistical analysis

When samples or data from more than one offspring from a litter were obtained, the data were averaged. All ‘n’ numbers refer to litters not individual animals. All data are presented as mean ± S.E.M. Statistical analysis was performed using multiple analysis of variance (ANOVA) followed by Dunnett's test. Unpaired Student's t test and ² test were also used when appropriate; P < 0.05 was considered significant.

	Results

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Maternal body weight and food intake

Maternal body weight at 10 days lactation was decreased in RR (P < 0.05; Table 2). Maternal food intake was not significantly altered by protein restriction during pregnancy but was lower at 10 days post-delivery in groups restricted in lactation (Table 2).

At 19 days of gestation maternal corticosterone, testosterone and oestradiol were elevated in the protein-restricted mothers (Fig. 1).

View larger version (11K): [in this window] [in a new window]   Figure 1.  Maternal serum corticosterone, testosterone and oestradiol in control and protein-restricted mothers at 19 d gestation Maternal serum (mean ± S.E.M.) corticosterone (ng ml–1), testosterone (ng ml–1) and oestradiol (pg ml–1) in control mothers fed the 20% casein diet (C; n = 10) and nutrient-restricted mothers (R; n = 10) fed the 10% casein diet; *P < 0.05.

There was no effect of diet on birth weight of the male pups. Pups of mothers fed the control diet weighed 6.33 ± 0.09 g and of restricted mothers 6.19 ± 0.11 g. However, ano-genital distance was increased by 12% as an absolute measure and 13% relative to body weight (P < 0.05; Fig. 2).

View larger version (7K): [in this window] [in a new window]   Figure 2.  Ano-genital distance of male offspring of rats fed control or restricted diet Ano-genital distance (mm), and ano-genital distance as mm g–1 of body weight at birth of male offspring of rats fed control (C – 20% casein) or restricted (R – 10% casein) diet during pregnancy. Mean ± S.E.M., n = 10–14 litters; *P < 0.01.

At 2 days postnatal age serum corticosterone in male pups of mothers fed the control diet was 167 ± 27.4 ng ml^–1 and 85 ± 3.1 ng ml^–1 in pups of protein-restricted mothers (P < 0.05).

Postnatal growth, timing of testicular descent, preputial separation and changes in testicular weight

Table 3 shows the body weights of the four groups of offspring at 25, 70 and 270 days PN. The body weight of RR and CR were significantly lower than the CC controls and the RC group at 25 and 70 days. At 270 days RR was 13% and CR 16% less than the CC group (P < 0.05).

At 25 and 70 days of age testicular weight was reduced in the two groups restricted during lactation (Table 4). At 270 days testis weight was reduced by 11% in RR and 6% in CR and RC but only the difference between RR and CC was significant. When expressed relative to body weight the only significant difference from CC was the relative testis weight in the CR group at 70 days (P < 0.05).

At 21 days PN, testosterone levels were 0.41 ± 0.01 ng ml^–1 (n = 5), 0.24 ± 0.06 ng ml^–1 (n = 4), 0.34 ± 0.07 ng ml^–1 (n = 6) and 0.53 ± 0.07 ng ml^–1 (n = 6) in CC, RR, CR and RC groups, respectively. There was a tendency for lower levels in the two groups whose mothers were restricted in lactation and levels in RR were lower than RC (P < 0.05). At 70 days PN serum LH was depressed in all three restricted groups compared with controls (Fig. 3). Testosterone demonstrated a similar pattern (Fig. 3). FSH levels did not differ between groups at 70 days. At 21 days old P450 scc mRNA was decreased in RR and CR (P < 0.05) but this difference did not persist to 70 days (Fig. 4).

View larger version (13K): [in this window] [in a new window]   Figure 3.  LH, FSH and testosterone serum concentrations at 70 days postnatal age in male offspring LH, FSH and testosterone serum concentrations at 70 days postnatal age in male offspring of rats fed with control (C – 20% casein) or restricted (R – 10% casein) diet during pregnancy (first letter) and lactation (second letter). Mean ± S.E.M., n = 4 litters; *different from CC, P < 0.01.

View larger version (25K): [in this window] [in a new window]   Figure 4.  Northern blot analysis of testis mRNA at 21 days (A) and 70 days (B) postnatal age Aa, P450scc mRNA; b, 28S ribosomal RNA; c, relative abundance of mRNA P450scc/ribosomal 28S in testes at 21 (A) and 70 (B) days postnatal age in male offspring of rats fed with control (C – 20% casein diet) or restricted (R – 10% casein diet) during pregnancy (first letter) and lactation (second letter). Mean ± S.E.M., n = 4 litters; *different from CC, P < 0.01.

Fertility rates were not different at 70 days of age but by 270 days, fertility had dropped by 50% in RC (Table 5: P < 0.05). Sperm count at this age was decreased in RR and RC (Table 5: P < 0.05).

	Discussion

Top Abstract Introduction Methods Results Discussion References

Our study was designed to determine separate and combined effects of protein restriction during the time windows of pregnancy and lactation. Although cross-fostering to non-biological mothers results in immediate exposure to the challenge under study, interpretation of this procedure must take into account considerations of immune and other differences compared with rearing by the biological mother. Keeping pups with their biological mother answers questions more directly relevant to human situations since future therapies to reverse effects of prenatal exposures will involve maintaining babies with their biological mother.

A major question in relation to protein restriction studies is the extent to which altered phenotypes are due to global protein restriction as distinct from deficiencies in individual micronutrients. We evaluated effects of an isocaloric, 50% protein diet on growth and sexual development of male rats. In our study, as in others, calories in the deficient diet were made up with carbohydrate. Thus protein-deprived mothers (and their fetuses) were exposed to a greater amount of carbohydrate which may act via effects on insulin and insulin-like growth factor production. We added cystine (as recommended by the American Nutrition Society) to the diets. To attain equivalence in cystine availability in proportion to overall protein, only half the amount of cystine was added to the protein-restricted diet compared with the control diet. The effects of the balance of amino acid availability in pregnancy are still poorly understood. For example, supplementation with glycine normalizes the hypertension seen in offspring exposed to some versions of 50% protein restriction (Jackson et al. 2002). Glycine supplementation also reverses the maternal endothelial dysfunction seen with the same diet (Brawley et al. 2004). Further studies, particularly of nutrient-dependent altered gene regulation will be necessary to provide a detailed explanation of the mechanisms involved. In this context, investigations need to address the varied diets consumed in different areas of the world. It is for this reason we used corn oil which is a major component of the diet in Mexico, rather than the soy used by other investigators (Reeves et al. 1993). Finally the effects of different fatty acid composition, especially different essential fatty acids, both their absolute amounts as well as the ratios, will need to be considered.

Effects on growth, sexual development and offspring endocrinology

The observed increase in ano-genital distance, an external marker of sexual differentiation at birth, following protein restriction in pregnancy in the absence of altered birth weight clearly indicates that normal birth weight can mask significant changes in key organ systems. Ano-genital distance is normally regulated by testosterone from the fetal testes and can be used as a marker of fetal exposure to endogenous fetal and transplacentally acquired maternal androgens (Graham & Gandelman, 1986). Since maternal steroids cross the placenta, the increase in maternal androgen observed at 19 days gestation may contribute to the increased ano-genital distance in pups of restricted mothers. Increased maternal glucocorticoid levels have been related to delay and decreased function in development of the sexual organs of male offspring (Page et al. 2001). The elevation of maternal corticosterone in the dams subjected to protein restriction would be anticipated to depress the fetal pituitary adrenal axis. This is a likely explanation for the lowered concentration of corticosterone observed in the 2 day old offspring. Other investigators using maternal decapitation under general anaesthesia did not observe an increase in maternal corticosterone with an isocaloric, 60% protein reduction (Fernandez-Twinn et al. 2003). These differences could be due either to a stress-induced increase in maternal pituitary adrenal axis function in our study or suppression by general anaesthesia in the other.

Determining the timing of the onset of puberty by external examination is difficult in the male. Precise definition of puberty requires evaluation of circulating LH changes. Prenatal exposure of fetal sheep to androgens has been shown to have clear effects on the LH changes that accompany puberty (Sharma et al. 2002) but there are no studies that report puberty-related endocrine changes in male rats to challenges during fetal life and lactation. Testicular descent and preputial separation are markers of sexual development. In our study, testicular descent was delayed in all three restricted groups and age at descent was not related to body weight. Our data provide no clear mechanism for this delayed testicular descent. However, the tendency to a lower testosterone at 21 days is compatible with the role of testosterone in regulating testicular descent (Shono et al. 1999; Shono & Suita, 2003). The delay in preputial separation, approximately 3 days, was not significant. Both intrauterine nutrient restriction produced by uterine artery ligation and during lactation by increasing litter size to 20, delay preputial separation unrelated to body weight (Engelbregt et al. 2000). Similarly preputial separation is delayed in offspring of mothers receiving 50% of global intake consumed by control mothers (Leonhardt et al. 2003).

In the adult it is well recognized that nutrient restriction will decrease gonadotropin production (Clarke & Henry, 1999). In our study LH and testosterone concentrations were reduced at 70 days postnatal age in offspring exposed to protein restriction at any developmental period, evidence for decreased pituitary–gonadal axis activity. We could not observe any changes in FSH at 70 days. It should be noted that using the same antibody FSH was decreased at weaning in the study of Leonhardt et al. (2003).

Adult function of the testis, fertility rate and sperm counts

Our findings agree with the view that exposure to high concentrations of maternal corticosterone adversely affect pituitary–gonadal and adrenal function in offspring. Over-exposure to glucocorticoids during development diminishes adult reproductive capacity shown in behaviours such as frequency of copulation and ejaculation (Stylianopoulou, 1983). Male rats exposed to prenatal stress show decreased sexual activity (Anderson et al. 1986).

There was a dissociation between inhibitory effects on testicular growth, which occurred following protein restriction during lactation, and sperm count which was impaired only by restriction during pregnancy. Feeding the control diet during the suckling period to mothers restricted during pregnancy normalized testicular growth and testosterone levels at 70 days. However, these pups (RC) were the only group to show both decreased sperm count and fertility. In contrast, the RR group had a lowered sperm count presumably as a result of restriction imposed during fetal life but fertility was unimpaired. These findings indicate the complexity of developmental programming effects on the multiple factors that regulate the entire process of fertility. It is generally considered that sperm are available in numbers greatly in excess of need for fertility. Prenatal restriction coupled with postnatal full nutrition may occur in the real world in the setting of placental insufficiency followed by adequate lactation. Continued protein restriction from fetal life up to weaning does not adversely affect fertility, thereby maintaining the ability of this species to procreate even in the face of shortage of nutrients. We hypothesize that the infertility that accompanies the unnatural switch from restriction before birth to plenty after birth represents an example of responses during fetal life which are maladaptive for the pups' postnatal nutritional status (Gluckman & Hanson, 2004).

Finally we have produced evidence that maternal protein restriction during development alters offspring testicular function at the biochemical level. Interestingly, changes in testosterone and P450scc mRNA did not always align. At 21 days testosterone levels in RR and CR pups tended to be lower while levels in RC were normal in keeping with significantly lower P450scc mRNA only in RR and RC at this age. However, by 70 days of life P450scc mRNA recovered to control levels in RR and CR despite the low testosterone in these groups. The explanation may lie in differences in regulation of secretion of progesterone or its differential use for synthesis and/or secretion of androgens between the groups.

In conclusion we have presented evidence for developmental programming effects at the pituitary and testicular level of the gonadal axis. We conclude that maternal protein restriction delays sexual maturation in male rats. It is clear that some effects emerge in later life. The finding that the major persistent effects on testicular growth resulted from maternal restriction in lactation agrees with the postnatal timing of the majority of cell division and growth in rats. The demonstration of postnatal effects on sperm numbers but prenatal effects on fertility emphasizes the importance of interactions between different key components of the testis in development.

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	Acknowledgements

 
This work was partially funded by CONACyT-138259 (Mexico) and the NIH HD21350.

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