Effects Of Dietary Protein Levels On Estrous Cycle And Follicular Development In Female Rats

Many studies showed that insufficient or excess protein intake may affect female estrous cycle and ovulation process, but the molecular mechanisms remained to be investigated further. This paper aimed to examine the effects of dietary protein levels on estrous cycle and follicular development in female rats, and the associated mechanisms. Female Sprague-Dawley (SD) rats with normal estrous cycle were randomly assigned to five diet treatments:standard diet containing 18% protein according to AIN-93 (CON, n=30), three protein restricted diets at 4%(PR4, n=30),8%(PR8, n=20) and 13%(PR13, n=20), protein excess diets at 23%(PE23, n=20). When the percentage of estrous cycle disturbed PR4 rats remained unchanged in eight days, ten irregular PR4 rats and ten normal CON rats were fed with CON diet to perform the re-feeding experiment, the remaining were sacrificed at the following proestrus to examine the estrous cycle and the related changes in tissue weight, ovarian morphology, metabolic hormone, reproductive hormone and amino acid levels, as well as gene expressions in hypothalamus and ovary. The re-feeding experiment was conducted until the PR4 rats showed two normal estrous cycles, then all rats were sacrificed for sample collection at the next proestrus. Results:1. Food intake and body weight:protein deficiency or excess did not affect food intake of rats (P>0.05). Body weight of PR4 rats was significantly lower at the end of 3 th, 4th,5th and 6th weeks compared with the other groups (P<0.05). Body weight of rats fed diets with protein content above 8% were not affected (P>0.05).1. Estrous cycle:during the 48 d experimental period, the average estrous cycle length of PR4 rats was significantly longer than the other groups (P<0.05), the estrous cycle number of PR4 and PR8 rat were significantly lower than the PR13, CON and PE23 rats (P<0.05), the proestrus, estrus, metestrus time of PR4 rats were significantly shorter than other groups, but diestrus time was significantly longer (P<0.05).3. Tissue weight and follicular development:ovary weight and ovary index of PR4 and PR8 rats were reduced compared with CON and PE23 rats (P<0.05), but fat pads index were higher (P<0.05). The absolute liver weight in PR4 rats was significantly lower than other rats (P<0.05). Rats fed PR4 diets showed a significant increase in the number of growing follicles and atretic follicles than other rats (P<0.05), but the number of antral follicles were the lowest (P<0.05).4. Serum hormone and amino acid levels:serum IGF-I, GnRH, LH and E2 levels were the lowest in PR4 rat (P<0.05). However, leptin and FSH levels were not affected (P>0.05). Among the essential amino acids, threonine, valine, leucine, phenylalanine levels of PR4 rats were decreased compared with CON rats (P<0.05). As to non-essential amino acids, serine, glutamate, glycine, alanine, citrulline, proline levels were elevated than CON rats (P>0.05). As the protein content varied from 4% to 23%, the ratio of total essential amino acid to total non-essential amino acid increased from 0.31 to 0.83.5. Gene expressions in hypothalamus and ovary:mRNA expressions of hypothalamic KiSS-1 in PR4 rats was significantly decreased compared with other rats (P<0.05), IGF-IR mRNA levels was significantly lower than PR13, CON and PE23 rats (P<0.05). mRNA expressions of ovary LHR and IGF-IR were significantly down-regulated in PR4, PR8 and PR 13 rats compared with CON rats (P<0.05). FSHR mRNA levels in PR4 rats was significantly lower than PE23 rats (P<0.05).6. Changes in parameters of the re-feeding experiment:PR4 rats experienced catch-up growth and cycle began to recover after eleven-day re-feeding by CON diets. Coincidently, gene expressions of hypothalamic KiSS-1, IGF-IR mRNA and ovary LHR and IGF-IR mRNA, as well as circulating IGF-I, GnRH, LH, E2 and amino acid concentrations were reversed to similar levels of that in CON rats (P>0.05).Conclusions:(1) 4% protein restriction extended estrous cycle length and impaired follicular development in sexually mature female rats.(2) Dietary protein could affect serum IGF-I concentration, which induced changes in hypothalamic IGF-IR and KiSS-1 mRNA expression, serum GnRH, LH, E2 levels, and the number of antral follicles, thus regulated the process of estrous cycles and follicular development in female rats.