The effects of letrozole-induced maternal hyperandrogenism on sexual behaviors, testicular histology, and serum biochemical traits in male offspring rats: An experimental study

Background: Intrauterine endocrine abnormalities have profound effects on the development of physiological disorders. Objective: This study aimed to assess the effects of in utero exposure to letrozole (an aromatase inhibitor) and its late consequences on the reproductive and metabolic performance of an adult male offspring. Materials and Methods: 15 pregnant Sprague-Dawley rats (8 wk, 155 gr) were randomly assigned into 5 experimental groups (n = 3/each) and orally received either letrozole at doses of 0.25, 0.75, 1.00, and 1.25 mg/kg body weight (BW) or vehicle (control) on the gestation days of 16, 17, and 18. Pregnancy outcome, sexual behaviors on postnatal day 60, serum biochemical features, and the histopathology of testes were assessed in male offspring. Results: Compared to control group, delayed labor (21.83 vs. 24.25, p < 0.0001) and reduced litter size (n = 12.25 vs. n = 2, p < 0.0001) were recorded in 1.25 mg/kg BW group. A reduction in high-density lipoprotein level and the elevation of testes weight, BW gain, anogenital distance, as well as the serum concentrations of testosterone, triglycerides, cholesterol, and glucose were observed in 1.25 mg/kg BW (p < 0.0001) and 1.00 mg/kg BW (p < 0.0001) groups in comparison to control. A larger number of anogenital female sniffing, pursuit, and mounting behaviors were also observed in 1.25 mg/kg BW group in comparison to control (p < 0.0001). Severe testicular defects including necrosis and disruption of the epithelium of seminiferous tubules, sloughing of epithelial cells, and spermatogenesis arrest were observed in letrozole-treated groups, in a dose-dependent manner. Conclusion: Maternal exposure to letrozole can adversely affect the reproductive and metabolic performance of male offspring rats, suggesting an incomplete sex differentiation. Key words: Androgens, Aromatase inhibitors, Rat, Sexual activities, Testes histopathology

Maternal aromatase inhibition via letrozole altered RFamide-related peptide-3 and gonadotropin-releasing hormone expression in pubertal female rats

Objective(s): Despite prevalence of polycystic ovary syndrome (PCOS) among childbearing women and development of many animal models for this syndrome, information on its etiology is still scarce. The intrauterine hyperandrogenic environment may underlie changes at the level of hypothalamus, pituitary, ovary organization in female offspring, and PCOS later in life. Letrozole has been shown to mimic reproductive and metabolic characteristics of PCOS in adult rodent models. Therefore, this research aimed to assess the condition in a prenatal letrozole-treated rat model. Materials and Methods: Twenty-eight female rats dams receiving letrozole at certain doses during late pregnancy were used in the trial. Pregnant Sprague-Dawley rats (n=21) received letrozole treatment on gestation days 16–18 at doses of 1.25, 1.0, 0.75, 0.5, and 0.25 mg/kg body weight (BW). Results: Prenatal letrozole treatment delayed parturition time and reduced the litter size in pregnant dams (P<0.0001). Late puberty onset, irregular ovarian cyclicity, increased anogenital distance (AGD), body weight gain, serum testosterone concentration, and reduced estradiol levels (P<0.0001) were observed in the female offspring of dams receiving 1.25 and 1 mg/kg BW letrozole. Furthermore, letrozole at 1.25 and 1 mg/kg BW showed increased RFRP and decreased GnRH mRNA expression (P<0.0001). Letrozole treatment at doses of 1 mg/kg BW and lower was not fetotoxic. Conclusion: It was concluded that 1 mg/kg BW letrozole may be suggested for prenatal PCOS induction. Keywords Gonadotropin-releasing hormone Hypothalamus Letrozole Polycystic ovary syndrome Prenatal Rat RFamide-related peptide-

The roles of RFamide-related peptides (RFRPs), mammalian gonadotropin-inhibitory hormone (GnIH) orthologues in female reproduction

Objective(s): To benefit from reproduction and deal with challenges in the environmental conditions, animals must adapt internal physiology to maximize the reproduction rate. Maladaptive variations in the neurochemical systems and reproductive system can lead to manifestation of several significant mammalian reprocesses, including mammalian ovarian lifespan. RFamide-related peptide (RFRP, Rfrp), mammalian orthologues of gonadotropin-inhibitory hormone (GnIH), which is a regulator to prevent the gonadotropin-releasing hormone (GnRH) neural activity, is known to be related to reproductive traits. This review aimed to summarize recent five-year observations to outline historic insights and novel perspectives into the functions of RFRPs in coding the mammalian reproductive physiology, especially highlight recent advances in the impact on RFRPs in regulating mammalian ovary lifespan.Materials and Methods: We reviewed the recent five-year important findings of RFRP system involved in mammalian ovary development. Data for this review were collected from Google Scholar and PubMed using the RFRP keyword combined with the keywords related to physiological or pathological reproductive functions.Results: Recent discoveries are focused on three major fronts in research on RFRP role in female reproduction including reproductive functions, energy balance, and stress regulation. The roles of RFRPs in various development phases of mammal reproduction including prepuberty, puberty, estrous cycle, pregnancy, milking, menopause, and/or ovarian diseases have been shown.Conclusion: Overall, these recent advances demonstrate that RFRPs serve as critical mediators in mammalian ovarian development

Ekspresija mRNA agutiju srodnog peptida i mRNA melanokortin-4 receptora u arkuatnoj jezgri za vrijeme gravidnosti i laktacije štakorica.

Pregnancy is associated with a range of physiological adjustments to adapt the body to the demands of the growth of the fetus and subsequent lactation. It has been observed that agouti-related peptide (AGRP) and melanocortin-4 receptor (MC4R) are involved in energy homeostasis. A randomized controlled experimental study was planned to investigate the expression of AGRP and MC4R mRNAs in the stages of pregnancy and lactation in rat arcuate nucleus (ARC) of the hypothalamus. Thirty-two adult female rats were randomly divided into six groups. Pregnant rats were assigned into three groups (n = 6) of 7, 14, and 21 days of pregnancy. Two more groups were also assigned of non-suckling rats (n = 5), immediately separated from their pups after parturition, and suckling rats (n = 5), allowed to suckle five pups until day 8 (increasing milk). The sixth group consisted of four ovariectomized rats, which were assigned two weeks after surgery and served as control. Using real-time PCR, the relative expressions (compared to controls) of MC4R and AGRP mRNAs in ARC were calculated in the pregnant, suckling and non-suckling rats. Expression of AGRP mRNAs in pregnant rats on days 14 and 21 was higher than that observed in suckling and non-suckling rats (P<0.05). Expression of MC4R mRNAs in pregnant rats on days 14 and 21 was lower than that observed in suckling rats, and was higher on day 7 than that observed in both suckling and non-suckling rats (P<0.05). In conclusion, expression of AGRP in pregnancy and MC4R in lactation in ARC of rats controls energy homeostasis.Gravidnost je povezana s nizom fizioloških prilagodbi kojima tijelo odgovara zahtjevima povezanima s rastom fetusa i kasnije laktacije. Uočeno je da su agutiju srodan peptid (AGRP) i melanokortin-4 receptor (MC4R) uključeni u energetsku homeostazu. Randomiziranim kontroliranim pokusom planirano je u arkuatnoj jezgri (ARC) hipotalamusa štakorica istražiti ekspresiju mRNA AGRP i mRNA MC4R tijekom gravidnosti i laktacije. Trideset dvije odrasle štakorice nasumično su bile podijeljene u šest skupina. Gravidne su štakorice podijeljene u tri skupine (n = 6), s obzirom na 7., 14. i 21. dan gravidnosti. Još dvije skupine (n = 5) činile su nedojne štakorice koje su odvojene od svoje mladunčadi odmah nakon porođaja te dojne štakorice kojima je dozvoljeno dojenje 5 mladunaca do osmog dana rastuće laktacije. Četiri ovarijektomizirane štakorice, dva tjedna nakon operacije, dodijeljene su u 6. kontrolnu skupinu. Koristeći PCR u stvarnom vremenu, relativna ekspresija (usporedba s kontrolama) mRNA MC4R i mRNA AGRP u ARC izračunata je za gravidne, nedojne i dojne štakorice. Ekspresija mRNA AGRP kod gravidnih štakorica 14. i 21. dan bila je veća od one opažene kod dojnih i nedojnih štakorica (P<0,05). Ekspresija MC4R mRNA u gravidnih štakorica 14. i 21. dan bila je manja u odnosu na dojne štakorica i veća 7. dan u odnosu na skupine dojnih i nedojnih štakorica (P<0,05). Zaključno, ekspresija AGRP u ARC tijekom gravidnosti i MC4R u ARC tijekom laktacije kontrolira energetsku homeostazu štakorica

Fast free of acrylamide clearing tissue (FACT) for clearing, immunolabelling and three-dimensional imaging of partridge tissues

Fast free of acrylamide clearing tissue (FACT) is a modified sodium dodecyl sulfate-based clearing protocol for the chemical clearing of lipids that completely preserves fluorescent signals in the cleared tissues. The FACT protocol was optimized to image translucent immunostained brain and non-nervous tissues. For this purpose adult male Chukar partridge (Alectoris chukar) was used as a model. After clearing the tissues, 1 or 2 mm-thickness sections of tissues were immunolabeled. The paraventricular nucleus in the hypothalamus (2-mm section) was cleared with FACT, and then was stained with gonadotropin-inhibitory hormone (GnIH) antibody and Hoechst. Simultaneously, immunohistochemical (IHC) staining of cryosectioned brain (30 μm) was done by GnIH-antibody. The FACT protocol and staining of cell nuclei of nine other tissues were done by a z-stack motorized fluorescent microscope. GnIH-immunoreactive neurons were found by FACT and IHC during the breeding season in male partridges. Deep imaging of the kidney, duodenum, jejunum, lung, pancreas, esophagus, skeletal muscle, trachea, and testis were also done. The FACT protocol can be used for the three-dimensional imaging of various tissues and immunostained evaluation of protein markers

The role of neuropeptides and neurotransmitters on kisspeptin/kiss1r-signaling in female reproduction

Reproductive function is regulated by the hypothalamic-pituitary-gonads (HPG) axis. Hypothalamic neurons synthesizing kisspeptin play a fundamental role in the central regulation of the timing of puberty onset and reproduction in mammals. Kisspeptin is a regulator of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). In female rodent, the kisspeptin (encoded by kiss1 gene), neurokinin B (Tac3) and dynorphin neurons form the basis for the “KNDy neurons” in the arcuate nucleus and play a fundamental role in the regulation of GnRH/LH release. Furthermore, various factors including neurotransmitters and neuropeptides may cooperate with kisspeptin signaling to modulate GnRH function. Many neuropeptides including proopiomelanocortin, neuropeptide Y, agouti-related protein, and other neuropeptides, as well as neurotransmitters, dopamine, norepinephrine and γ-aminobutyric acid are suggested to control feeding and HPG axis, the underlying mechanisms are not well known. Nonetheless, to date, information about the neurochemical factors of kisspeptin neurons remains incomplete in rodent. This review is intended to provide an overview of KNDy neurons; major neuropeptides and neurotransmitters interfere in kisspeptin signaling to modulate GnRH function for regulation of puberty onset and reproduction, with a focus on the female roden

Pathophysiological mechanisms of gonadotropins- and steroid hormones-related genes in etiology of polycystic ovary syndrome

Objective(s): Polycystic ovary syndrome (PCOS) is an endocrinopathy in women, which, unlike its impact on fertility and health of women, there is no clear understanding about the causal mechanisms of this pathogenesis. The aim of this review paper is to investigate the pathophysiological pathways affecting the PCOS etiology, based on functions of gonadotropins– and steroid hormones–related genes. Materials and Methods: Due to different hormonal and metabolic signs of this complex disorder, different hypotheses are mentioned about etiology of this syndrome. Because of the heterogeneity of the reasons given for this syndrome and the spread of the effective genes in its pathophysiology, most of genes affected by sex-related hormonal imbalances are examined for discriminative diagnosis. For this purpose, published articles and reviews dealing with genetic evaluation of PCOS in women in peer-reviewed journals in PubMed and Google Scholar databases were included in this review. Results: In previous studies, it has been well demonstrated that PCOS in some individuals have a genetic origin. Pathophysiological functions of genes are primarily responsible for the synthesis of proteins that have role in PCOS before hyperandrogenism including GnRHR, FSHβ, FSHR, LHCGR, CYP19A1, HSD17B, AR and SHBG, and their effects in PCOS of human have been confirmed. Conclusion: Hormonal imbalances are the first reason mentioned in PCOS etiology, and usually characterized with menstrual irregularities in PCOS women. Hyperandrogenism and gonadotropin secretion disorders are shown in PCOS condition, which are related to steroidogenesis pathways and hypothalamic–pituitary–ovarian axis disturbances, respectively

Expression of melanocortin-4 receptor and agouti-related peptide mRNAs in arcuate nucleus during long term malnutrition of female ovariectomized rats

Objective: Melanocortin-4 receptor (MC4R) and agouti-related peptide (AgRP) are involved in energy homeostasis in the rat. The aim of the present study was to evaluate the expression of MC4R and AgRP mRNAs in arcuate nucleus (ARC) during long term malnutrition of female ovariectomized rats. Materials and Methods: Ten female ovariectomized rats were divided into two equal groups (n=6) of normal and restricted diet groups. Using real-time PCR, the relative expressions (compared to controls) of MC4R and AgRP mRNAs were compared between both diet groups. Results: The relative expression of MC4R and AgRP mRNA in the ARC of female ovariectomized rats during long term malnutrition was higher than those with normal diet (