Influence Of Prepubertal Exposure To Low Dose Of Bisphenol A On Sexual Development Of Male Rats And Its Underlying Mechanisms

BackgroundEndocrine disrupting compounds (EDCs) are chemicals in the environment thatmimic or inhibit the actions of endogenous hormones, and have the potential to alterthe structure and function(s) of the endocrine system. Bisphenol A (BPA), anenvironmental estrogenic chemical, has been attracting increased attention because ofits high potential for human exposure. Multiple laboratories have shown that neonatalor perinatal exposure to BPA, at doses even lower than the “safe” exposure limit forhumans, can affect the onset of puberty and sexual maturation in rodents. In malerodents, exposure of fetus or neonate to environmentally relevant concentrations ofBPA brings about a high incidence of reproductive disorders such as cryptorchidism,decreased sperm count, and erectile dysfunction. Neonatal exposure of rats to BPAcan alter the structure and function of testis and reproductive tract to advance the firstwave of spermatogenesis at puberty. Puberty development is under control ofdifferent hormonal regulations. Administration of exogenous hormones such asestradiol (E2) and testosterone to pubertal rodents disrupts the progression of puberty,indicating that puberty is an impressionable window for estrogenic regulations.However, to date, little is known concerning the impact of prepubertal exposure toBPA on the male reproductive development.Hypothalamic-pituitary-gonadal (HPG) hormones are very important for onsetand maturation of sexual development. The activation of gonadotropin-releasinghormone (GnRH) neurons in the basal forebrain is critical step in the pubertal onset ofHPG axis. Since GnRH neurons do not express estrogen receptor α (ERα), theyaccept gonadal steroid signaling largely from ERα-expressing kisspeptin neuronslocated at the arcuate nucleus (ARC) in males. At puberty, kisspeptin binding with G protein-coupled receptor54(GPR54) can induce the activation of GnRH neurons.Recently, the kisspeptin neurons in ARC have been identified to express neurokinin-B(NKB) and dynorphin (Dyn), abbreviated as KNDy neurons. Either KNDy neurons orGnRH neurons have been known to express the Dyn receptors and KNDy receptors.The previous studies reported that NKB and Dyn could regulate the activities ofKNDy neurons and GnRH neurons. Several recent studies provide tantalizing-albeitindirect-evidence that the KNDy neurons serve as the central pacemaker to drivepulsatile GnRH and LH secretion. KNDy neurons express ERα that mediates negativefeedback of E2in the rhythmic discharge of KNDy neurons and the inhibition ofKiss1expression in ARC. The E2-inhibited kisspeptin expression would be expectedto reduce the secretion of GnRH pulse.Leydig cells produce the primary male steroid hormone testosterone thatstimulates the development of testis and reproductive tract in puberty and regulatesspermatogenesis and fertility in adulthood. The gonadotropin LH controlstestosterone (T) secretion from Leydig cells. The change in plasma level of thegonadotropin (LH and FSH) or T can influence the development of testicle. Inaddition, BPA has been reported to alter the production and secretion of T. Thepresent study focused on exploring (1) Impact of prepubertal exposure to anenvironmental dose of BPA on the pubertal onset of HPG axis and the development oftesticle in male rats;(2) the mechanisms of reproductive endocrine and cellulardamage underlying prepubertal treatment with BPA to influence development oftesticle.Objective1. Impact of prepubertal exposure to low dose of BPA on pubertal onset of HPG axisand its mechanisms;2. Influence of prepubertal exposure to low dose of BPA on development of testicleand underlying mechanisms.—The First Part Impact of prepubertal exposure to low dose of BPA on pubertal onset of HPG axisand its mechanismsMaterials and Methods1. Preparation of animal models: postnatal day21(PND21) male rats were givensubcutaneous administration of BPA (2μg/kg/day) or E2(0.4μg/kg/day) for14days. PND35BPA-rats, PND35E2-rats, or in20days after the cessation of BPAPND55BPA-rats were used.2. The levels of serum LH, FSH and T were measured by radioimmunoassay.3. Kisspeptin and GnRH neurons were examined by immunohistochemistry.4. Levels of Kiss1, NKB and Dyn mRNA in ARC, and GnRH mRNA in POA wereexamined by RT-PCR.Results1. In comparison with controls, the serum level of T reduced, whereas the levels ofLH and FSH increased in PND35BPA-rats. Either the levels of LH and FSH orlevel of T decreased in PND35E2-rats. In PND55BPA-rats, the levels of LH andFSH had no significant difference between control and BPA-rats, but the level ofT reduced2. In PND35BPA-rats, the number of GnRH immunoreactive (GnRH+) cells had nodifference from control rats, the level of GnRH mRNA elevated. By contrast thenumber of GnRH+cells and the level of GnRH mRNA were lower in PND35BPA-rats compared to control levels.3. In ARC either the number of kisspeptin immunoreactive (kisspeptin+) cells, or thelevels of Kiss1, NKB and Dyn mRNA were higher in PND35BPA-rats than thosein control rats. In PND35E2-rats, the number of kisspeptin+cells, and the levelsof Kiss1and NKB mRNA in ARC significantly decreased, which was partiallyprevented by the treatment with BPA.4. The frequency of LH-pulses in BPA-rats increased approximately15%of controllevel, whereas its amplitude was not altered. The frequency of LH-pulses had nosignificant difference between control and E2-rats; however its amplitude was lower in E2-rats.ConclusionThe results indicate that the prepubertal exposure of male rats to low dose of BPAcan enhance the activation of KNDy neurons to enhance the GnRH/LH secretion,while it inhibits the production of T.—The Second PartInfluence of prepubertal exposure to low dose of BPA on development of testicle andunderlying mechanismsMaterials and Methods1. Histological examination of testis: Body weight (BW), testicle weight (TW), testisindex (TW/BW), diameter and epithelium height of seminiferous tubules wereexamined. Seminiferous tubules with elongating spermatids and germ cells atstage I-VIII in PND35rats were examined. Sperm counts and germ cells at stageVII-VIII in PND55rats were examined.2. Apoptotic index of germ cells was examined by TUNEL staining.3. Levels of Fas, FasL, caspase3mRNA in testis were examined by RT-PCR.Results1. In PND35BPA-rats BW was increased compared to controls, but the TW and testisindex had no significant difference between both. BW and TW had no significantdifference between PND55control and BPA-rats.2. The diameter and epithelium height of seminiferous tubules in PND35and PND55BPA-rats were apparently reduced compared to control rats.3. In comparison with controls, the numbers of spermatogonia and round spermatidsin stage I-VIII in PND35BPA-rats, the number of round spermatids in stageVII-VIII in PND55BPA-rats were reduced.4. The apoptotic index (percent of seminiferous tubules containing more than three apoptotic germ cells) was higher in PND35BPA-rats, but not PND55BPA-rats,than that in control rats.5. The levels of Fas, FasL, caspase3mRNA in testis were increased in PND35BPA-rats compared to control rats, but in PND55BPA-rats.ConclusionThe results indicate that the prepubertal exposure of male rats to low dose of BPAimpaired testicle development via reducing the level of testosterone to inhibit theprocess of meiosis and cascading Fas/FasL signaling to induce germ cell apoptosis.