Preputial Separation Research Articles

7788 Importance of Neuronal STAT3, but Not ERK2 or mTOR, for Pubertal Timing and Reproductive Activity in Mice

Abstract Disclosure: R.A. Lord: None. M.A. Inglis: None. G.M. Anderson: None. The adipose-derived hormone leptin is important for regulating reproduction. The canonical JAK2/STAT3 pathway is the best-characterised leptin receptor signalling pathway. Neural STAT3 deletion is known to cause obesity; however, its reproductive influence is less understood. This experiment investigated whether STAT3 knockout from brain neurons or AgRP neurons, a neuronal population necessary for normal pubertal timing and reproduction, would unveil the necessity of STAT3 in reproductive function. We also tested the role of ERK2 and mTOR, two alternative leptin signalling molecules.Transgenic mice with a neuronal knockout were created via the Cre-loxP system. CamKinaseIIα-Cre mice were crossed with STAT3, mTOR or ERK2 floxed mice. AgRP-Cre mice were crossed with STAT3 floxed mice for STAT3 knockout in AgRP neurons. All groups were compared to Cre-negative littermate controls (n=6-12/group). Puberty onset was assessed post-weaning by examining genitalia development. Reproductive cyclicity and organ weights were measured in adults. Metabolic effects were evaluated through body weight, abdominal fat and fasting glucose measurements. Brain tissue analysis evaluated cellular responses to leptin for STAT3, ERK2, and mTOR. Data presented as mean ± SEM.Mice with ERK2 KO or mTOR KO showed no reproductive or metabolic deficits compared to controls. In marked contrast, neuronal STAT3 KO mice showed significantly increased body weight by 5 weeks of age and a 6-fold increase in abdominal adiposity as adults (both p<0.001 using two-way ANOVA and Student’s t-test respectively) compared to controls. Males had a significant 5-day delay in age at preputial separation (KO: 32.1±2.09 d; Control: 26.8±0.45 d, p<0.01, Student’s t-test) while females had a significant 7-day delay in vaginal opening (KO: 33.0±1.85 d, Control: 26.6±0.37 d, p<0.05, Student’s t-test), a 9-day delay in first estrus (KO: 37.8±1.89 d; Control: 29.4±0.65 d, p<0.01, Student’s t-test) and pronounced acyclicity. Neuronal STAT3 KO mice also had a >2-fold elevation in fasting glucose levels (p<0.001, Student’s t-test) and regressed reproductive organs. Female mice with STAT3 knockout in AgRP neurons showed significantly increased body weight (by 3 weeks [p<0.001], two-way ANOVA) and a significant 3-day delay in first estrus (KO: 31.1±0.71 d, Control: 28.6±0.45 d, p<0.01, Student’s t-test). These data show that neuronal STAT3 is critical for correctly timed puberty and subsequent fertility in male and female mice, whereas mTOR and ERK2 are not essential. These results have prompted a re-evaluation of previous conclusions about the role of STAT3 from experiments using LepR-Cre mice. Additionally, AgRP neuronal requirements for STAT3 exactly recapitulate AgRP requirements for leptin receptors. A better grasp of leptin signalling pathways will improve our understanding of how infertility arises due to metabolic disease. Presentation: 6/3/2024

8134 Hypothalamic Dlk1 Expression is Not Essential for Preventing Early Maturation of the Reproductive Axis in Female and Male Mice

Abstract Disclosure: D.B. Macedo: None. H. Kim Kyeol: None. A. Abreu: None. A. Latronico: None. R.S. Carroll: None. U.B. Kaiser: None. Background and Objectives: Inactivating mutations in Delta-like homolog 1 (DLK1), a maternally imprinted gene on chromosome 14, have been recognized as a genetic cause of central precocious puberty (CPP) in humans. It is well established that the timing of puberty, especially in females, is highly sensitive to energy stores and metabolic cues, with lower body fat negatively correlated with age at puberty and menarche. Mice lacking Dlk1 have pre- and postnatal growth retardation. Despite their considerably lower body weight (BW), we found that Dlk1 deficient females achieved puberty at the same age as controls (‘relative precocious puberty’). Moreover, Dlk1 deficiency led to activation of the reproductive axis despite lower levels of kisspeptin and leptin, an adipose tissue hormone with a permissive/stimulatory effect on metabolic control of reproduction. Dlk1 is a transmembrane protein that plays an essential role in inhibiting adipocyte differentiation and its biologically active form is soluble. Increased hypothalamic expression postnatally suggests a neuroendocrine function, but the precise mechanism by which DLK1 deficiency leads to CPP is yet to be deciphered. In this study, we aimed to investigate the role of hypothalamic Dlk1 expression on pubertal onset without the interference of diminished BW, using a conditional knockout (cKO) mouse model. Methods and Results: We generated a targeted Dlk1 knockout mouse model by crossing mice expressing Cre recombinase under the control of Nestin gene (Nes Cre), expressed primarily in neuro-epithelial cells, with mice harboring the Dlk1 gene flanked by loxP sites (Dlk1fl). Since Dlk1 is imprinted and expressed only by the paternal allele, we bred heterozygous Dlk1fl/+ male mice with Nes Cre females to obtain Dlk1fl/Cre (Dlk1 cKO), Dlk1+/Cre (Nes Cre) and Dlk1+/+ (wild type - WT). We confirmed by RT-qPCR that Dlk1 mRNA was undetectable in the mediobasal hypothalamus of Dlk1 cKO adult male mice, whereas it was present in Nes Cre and WT mice. We noted a lower BW in Nes Cre compared to WT mice, as previously reported. However, no difference in BW was observed between Dlk1 cKO and Nes Cre mice at the timing of puberty (females: Dlk1 cKO 12.6 ± 0.8 g vs. Nes Cre 13.8 ± 0.2 g, P = 0.18; males: Dlk1 cKO 12.4 ± 0.2 g vs. Nes Cre 13.1 ± 0.5 g, P = 0.26). Puberty onset, assessed by age at vaginal opening (females) and at preputial separation (males), was not affected by hypothalamic Dlk1 deletion (Dlk1 cKO: 35.6 ± 2.7 days vs. Nes Cre 34.6 ± 0.7 days, P = 0.73; Dlk1 cKO 29.0 ± 0.6 vs. Nes Cre: 30.3 ± 0.6 days, P = 0.19). Conclusion: In contrast to global Dlk1 deficiency, targeted deletion of Dlk1 from neuronal and glial cells did not affect body weight or timing of pubertal onset. These findings suggest that Dlk1 originates from peripheral tissues, such as adipocytes, to regulate reproduction and metabolism. Presentation: 6/1/2024

P-638 Paternal Consumption of Caloric and Non-Caloric Sweeteners is Associated with Sex-Specific Differences in Reproductive outcomes in offspring

Abstract Study question Does paternal pre-conception consumption of caloric or non-caloric sweeteners (NCS) coupled with high fat diets (HFD) predispose offspring to early puberty onset in mice? Summary answer Paternal prenatal consumption of the NCS Rebaudioside A(RebA) resulted in early puberty onset in female offspring only compared to water and caloric sweetener groups. What is known already A growing body of evidence suggests that the paternal diet also influences health and disease onset in offspring. Early puberty onset is associated with metabolic syndrome, type 2 diabetes, and cardiovascular disease later in adulthood. NCS are often promoted as heathier alternatives to caloric sweeteners. Evidence examining the impact of NCS on male reproductive function and offspring health outcomes, including pubertal timing are lacking. Few studies have shown maternal HFD can contribute to early puberty onset in female offspring however to our knowledge this is the first study to investigate paternal diet and offspring reproductive outcomes. Study design, size, duration 32 6-week-old C57/BL6 male mice were randomly assigned to receive a control diet (CD) or HF plus water (Con), fructose (Fr), Acesulfame K (Ak) or RebA). After 8 weeks, mice were mated. Litters were standardised to 2 males and 2 females per group and fed a standard laboratory diet. Participants/materials, setting, methods Puberty onset was measured in females by vaginal opening (from postnatal day (PN28) and in males by preputial separation (from PN35). Oral glucose tolerance tests were carried out at PN70. Mice were culled from PN80. Data were analysed by repeated measures or 2-way ANOVA. Main results and the role of chance There were no significant differences in birth weight, sex ratio or organ weights across groups. There was an overall effect of paternal NCS on male but not female offspring weight[CR1] at cull(p < 0.05), with male CDRebA offspring having increased BW (29.52g±1.5) compared with CDAk at PN80 (26.4g±3; p < 0.05). Females from paternal HF and CD RebA group [CR2] entered puberty significantly earlier (31.8d±1) than Fr (34d±1;p<0.01) and Con (34±1;p<0.05) groups. Male offspring from HFAk had lower glucose AUC (913±31) compared with CDCon (1289±48;p<0.001). Female offspring from CDRebA (1263±84) and CDFr (1273±67) groups had lower glucose AUC compared with CDCon group (1445±56;p<0.05). Limitations, reasons for caution This study was carried out on mice therefore direct translatability to humans is limited. examining the impact of paternal NCS intakes on molecular pathways in the ovary and testes to assess offspring reproductive health later in life and to evaluate pregnancy success following mating would essential for providing further insight. Wider implications of the findings The present study adds to the experimental evidence to date suggesting that NCS may not be beneficial alternatives to sugar-sweetened products consumed preconceptionally on the health of the offspring. This study also highlights the importance of including paternal diet in research when looking at transgenerational effects. Trial registration number not applicable

Can exposure to lisdexamfetamine dimesylate from juvenile period to peripubertal compromise male reproductive parameters in adult rats?

Lisdexamfetamine (LDX) is a d-amphetamine prodrug used to treat attention deficit and hyperactivity disorder, a common neurodevelopmental disorder in children and adolescents. Due to its action mediated by elevated levels of catecholamines, mainly dopamine and noradrenaline, which influence hormonal regulation and directly affect the gonads, this drug may potentially disrupt reproductive performance. This study evaluated the effects of exposure to LDX from the juvenile to peripubertal period (critical stages of development) on systemic and reproductive toxicity parameters in male rats. Male Wistar rats (23 days old) were treated with 0; 5.2; 8.6 or 12.1 mg/kg/day of LDX from post-natal day (PND) 23 to 53, by gavage. LDX treatment led to reduced daily food and water consumption, as well as a decrease in social behaviors. The day of preputial separation remained unaltered, although the treated animals exhibited reduced weight. At PND 54, the treated animals presented signs of systemic toxicity, evidenced by a reduction in body weight gain, increase in the relative weight of the liver, spleen, and seminal gland, reduction in erythrocyte and leukocyte counts, reduced total protein levels, and disruptions in oxidative parameters. In adulthood, there was an increase in immobile sperm, reduced sperm count, morphometric changes in the testis, and altered oxidative parameters, without compromising male sexual behavior and fertility. These findings showed that LDX-treatment during the juvenile and peripubertal periods induced immediate systemic toxicity and adversely influenced reproductive function in adult life, indicating that caution is necessary when prescribing this drug during the peripubertal phase.

Parameatal Urethral Cyst in a 20-Year-Old Male Patient: A Case Report

The parameatal urethral cyst is a rare anomaly and appears as a small blister in proximity to the urethral meatus. These cysts may result from occlusion of paraurethral ducts or, in other cases, from faulty preputial separation from the glans along the coronal sulcus. A 20-year-old male patient came to our outpatient clinic at dr. M. Soewandhie General Hospital Surabaya with a cystic lesion on the right side of the external urethral meatus. The patient only found out about his situation one year ago, and he did not experience any complaints except a branched urination. On physical examination, an 8 mm cystic lesion was found on the right side of the external urethral meatus. The patient consented to complete surgical excision of the cyst. The procedure was performed under regional anesthesia. The cyst was completely resected from the surrounding tissues. One month after surgery, there is no post-operative pain and good urine flow, and the patient is also satisfied with the post-operative condition. Histologic examination shows pieces of tissue in the form of polypoid lined with squamous epithelium underneath, stroma of fibrous tissue with proliferation of blood vessels and some inflammatory cells of lymphocytes in between. There are no signs of malignancy. Parameatal urethral cyst is an abnormal and rare case, usually asymptomatic. However, sometimes dysuria, urinary retention, branching urine, and cosmetic problems are the reasons that cause patients to seek a doctor's help. It can be diagnosed just on physical examination and can be cured by complete excision.

Prepubescent Electronic Cigarette Exposure Affects Sexual Motivation and Puberty in Female But Not Male Long-Evans Rats.

A method for delivering vaporized nicotine to animals has been developed using e-cigarette devices. The present experiment was designed to measure the effects of e-cigarette nicotine on pubertal onset and development of reproductive behavior in female and male Long-Evans rats. Rats received daily 10-min sessions of electronic-cigarette vaporized nicotine (5% Virginia Tobacco JUUL Pods) or room air in a whole-body exposure chamber (postnatal day 28-31). Pubertal onset was monitored daily (ie, vaginal opening in females, preputial separation in males). Two weeks later, rats were tested for sexual motivation using the partner-preference paradigm, whereby subjects were given the opportunity to approach either a sexual partner or a same-sex social partner. Four weeks later, partner preference was assessed again, 10 min after rats were re-exposed to their same prepubertal treatment. We found that prepubescent electronic-cigarette vaporized nicotine disrupted puberty and sexual motivation in female but not male rats. In vaped females, vaginal opening was delayed and less time was spent with the male stimulus compared to room-air controls. In contrast, no effect of e-cigarette vapor was observed on pubertal onset or on any measures of sexual behavior in male rats. No effects were observed in either female or male rats on the second partner-preference test. Prepubescent vaporized nicotine affected the development of reproductive physiology and behavior in female rats but not in male rats, whereas an additional acute exposure to nicotine vapor had no effect in either female or male adult rats. Given the prevalence of increasingly younger users, more animal research is needed to explore the effects of e-cigarette smoking on multiple developmental systems including reproductive physiology and behavior. This model could be useful in exploring multiple behavioral and physiological endpoints in both sexes. Adjustments to the duration of exposure and control conditions will be necessary for future experiments to best model human use.

Sleep deprivation alters pubertal timing in humans and rats: the role of the gut microbiome.

Evidence implied that sleeping duration is associated with the timing of puberty and that sleep deprivation triggers early pubertal onset in adolescents. Sleep deprivation can affect metabolic changes and gut microbiota composition. This study investigated the effects of sleep deprivation on pubertal onset and gut microbiota composition in animal models and a human cohort. This study comprised 459 boys and 959 girls from the Taiwan Pubertal Longitudinal Study. Sleep duration was evaluated using the self-report Pittsburgh Sleep Quality Index questionnaire. Early sexual maturation was defined by pediatric endocrinologist assessments. Mediation analyses were done to examine the association between sleep parameters, obesity, and early sexual maturation. Besides, Sprague Dawley juvenile rats were exposed to 4 weeks of chronic sleep deprivation. Vaginal opening (VO) and preputial separation (PS) were observed every morning to determine pubertal onset in female and male rats. The sleep-deprived juvenile rats in the sleep-deprived-female (SDF) and sleep-deprived-male (SDM) groups experienced delayed VO (mean VO days: 33 days in control; 35 days in SDF; p-value < 0.05) and PS (mean PS days: 42 days in control; 45 days in SDM; p-value < 0.05), respectively. Relative to their non-sleep-deprived counterparts, the sleep-deprived juvenile rats exhibited lower body weight and body fat percentage. Significant differences in relative bacterial abundance at genus levels and decreased fecal short-chain-fatty-acid levels were identified in both the SDF and SDM groups. In the human cohort, insufficient sleep increased the risk of early sexual maturation, particularly in girls (OR, 1.44; 95% CI: 1.09 to 1.89; p-value < 0.01). Insufficient sleep also indirectly affected early sexual maturation in girls, with obesity serving as the mediator. Overall, sleep deprivation altered the timing of puberty in both animal and human models but in different directions. In the rat model, sleep deprivation delayed the pubertal onset in juvenile rats through gut dysbiosis and metabolic changes, leading to a low body weight and body fat percentage. In the human model, sleep deprivation led to fat accumulation, causing obesity in girls, which increased the risk of early puberty.

Determinants of puberty and age of sexual maturity in Nigerian indigenous breeds of male goats

This study was conducted to ascertain the pubertal and sexual maturity characteristics of Nigerian native goat breeds. The research was carried out on three goat breeds; Sahel (SG), Red Sokoto (RSG), and West African Dwarf (WAD) goats. Each breed was represented by eight (8) animals (n=24) and housed in separate pens based on breed type. All bucks were weighed at birth and bi-weekly using digital weighing scales until they reached puberty. Scrotal circumference measurements were taken every two weeks until the end of the study. Semen was collected immediately after preputial separation and all semen samples were labelled and placed in a thermo-flask immediately and subsequently transported to the laboratory for evaluation. The mass motility and the forward motilities of the spermatozoa were observed using a light microscope. The percentage of live, dead, and abnormal sperms were determined after staining with eosin and negrosin stains. The sperm cell concentration was determined by counting spermatozoa with a hemocytometer. Result obtained indicate that at the 14th and 16th weeks of age, the mean scrotal circumference of bucks ranged from 5.14±0.66 cm to 7.06±0.71 cm, 6.32±0.29 cm to 8.05±0.42 cm, and 5.50±0.74 cm to 7.06±0.71 cm in SG, RSG, and WAD bucks, respectively. Puberty in Red Sokoto bucks was achieved at an average age of 14 weeks, with a mean sperm concentration of (116.17±28.47 x 106), progressive sperm motility of (65.00±9.92%), and a mean body weight of 10.17±1.26 kg. Sahelian and WAD bucks reached puberty at the 16th weeks, with sperm concentrations of 106.29±25.18 x 106 and 107.60±19.99 x 106, mean sperm motility of 65.00±9.94% and 76.00±4.8%, and mean body weights of 11.29±1.59 kg and 7.78±0.70 kg, respectively. To summarise, this study revealed that breed, age, scrotal circumference and body weight at puberty played significant roles in determining sperm characteristics and sexual maturity.

SAT608 Gonadotrope-specific Deletion Of Icer Results In Increased Lh Pulsatility But Reduced Fsh Levels In Mice

Abstract Disclosure: N.P. Boris: None. S.A. Pereira: None. G.A. Stamatiades: None. K.N. Hausken: None. H. Kim: None. R.S. Carroll: None. U.B. Kaiser: None. Background: The hypothalamic-pituitary-gonadal (HPG) axis is regulated by pulsatile secretion of hypothalamic GnRH, which acts on anterior pituitary gonadotropes to control synthesis and secretion of LH and FSH. High and low GnRH pulse frequencies preferentially stimulate Lhb and Fshb expression, respectively, through mechanisms that are not completely understood. The mouse Fshb gene promoter contains a half-AP-1/half-cAMP response element (CRE) motif; we have shown previously that this element is bound by CRE-binding protein (Creb) in vitro to increase expression in response to low GnRH pulse frequencies, but is antagonized by inducible cAMP early repressor (Icer) at higher GnRH pulse frequencies, resulting in a lower level of Fshb transcription. In this study, we aimed to identify the role of Icer in gonadotropin regulation in vivo in mice. We hypothesized that gonadotrope-specific deletion of Icer may influence pubertal timing and fertility due to an increase in Fshb expression. Methods and Results: Gonadotrope-specific deletion of Icer (GnrhrGRIC/+/Icerfl/fl; Icer KO) and control (Icerfl/fl) mice were generated, and pubertal and reproductive phenotypes were evaluated. There was no difference in body weight or in pubertal timing, evaluated by age of preputial separation in males and by vaginal opening and first estrus in females. In post-pubertal male mice, neither basal LH and FSH levels nor the LH or FSH response to acute GnRH stimulation were significantly different in Icer KO mice, compared to littermate Icerfl/fl controls. Male Icer KO mice 14 days post-gonadectomy (GDX) had significantly more LH pulses within a three-hour period than controls (Icer KO 5.83 ± 0.4 pulses vs Icerfl/fl 4.66 ± 0.3 pulses; p=0.049), but no differences in basal LH or LH pulse amplitude. Interestingly, serum FSH levels were significantly lower in Icer KO mice compared to Icerfl/fl controls 2 days post-GDX (Icer KO 20.0 ± 1.0 vs Icerfl/fl 26.0 ± 0.8; p=0.003) and 7 days post-GDX (Icer KO 18.0 ± 0.4 vs Icerfl/fl 22.5 ± 0.8, p=0.002). There were no significant differences in pituitary Fshb, Lhb, or Gnrhr mRNA levels between Icer KO and Icerfl/fl control mice 14 days post-GDX. Conclusions: Deletion of Icer from pituitary gonadotropes in mice did not affect body weight or timing of pubertal onset. However, in GDX male mice, LH pulse frequency was increased, whereas the post-GDX increase in serum FSH levels was blunted, in Icer KO mice compared to controls. These findings support a role for Icer in both LH and FSH regulation in vivo and highlight the importance of extending in vitro observations to in vivo models. Presentation: Saturday, June 17, 2023

THU190 Deletion Of The Glucocorticoid Receptor From GABAergic Neurons Causes Early Puberty In Male And Female Mice

Abstract Disclosure: S.A. Pereira: None. L. Arvizu-Sanchez: None. A.N. Fontes: None. R.S. Carroll: None. V.M. Navarro: None. U.B. Kaiser: None. Background and Objectives: Glucocorticoid (GC) secretion induced by various types of stress can negatively impact reproduction through effects at several levels of the hypothalamic-pituitary-gonadal (HPG) axis, including inhibition of gonadotrophin-releasing hormone (GnRH) secretion and suppression of luteinizing hormone (LH) release. KNDy neurons, expressing kisspeptin, neurokinin B and dynorphin A, are the GnRH and LH pulse generator and are key regulators of puberty. These neurons are proposed to play an important role in mediating the effects of stress and GC on reproduction. While the precise mechanisms by which stress affects regulation of KNDy neurons remain to be fully characterized, it is known that KNDy neurons are subject to regulation by multiple neural networks, among which GABAergic neurons appear to play a determinant role. Importantly, a large proportion of GABAergic neurons express glucocorticoid receptors (GR). We therefore hypothesized that stress, through the action of GC on GR in GABAergic neurons, affects the activity of KNDy neurons to impact puberty timing. Methods and Results: We generated mice with conditional deletion of GR in GABAergic neurons using VgatCre mice crossed with GRlox/lox mice to generate VgatCre;GRlox/lox mice (GABAGRKO). The pubertal phenotypes of these GABAGRKO mice and their littermate controls (GRlox/lox) were characterized. Male GABAGRKO mice had earlier preputial separation compared to GRlox/lox littermate controls (GABAGRKO 29.8 ± 0.45 days vs. GRlox/lox 33.5 ± 0.57 days; p &amp;lt; 0.0001). Female GABAGRKO mice had a similar age of vaginal opening (GABAGRKO 25.7 ± 0.76 days vs GRlox/lox 24.5 ± 0.87 days; p = 0.2810) but had significantly earlier first estrus compared to their GRlox/lox littermate controls (GABAGRKO 32.4 ± 0.76 days vs GRlox/lox 37.9 ± 0.79 days; p &amp;lt; 0.0001). Male and female mice were euthanized at 30 days of age and their mediobasal hypothalamus (MBH) were collected for mRNA measurement by RT-qPCR of genes known to be related to pubertal development, including Kiss1, Tac2, Pdyn, Agrp and Pomc. Kiss1 and Pdyn expression were significantly higher in the MBH of GABAGRKO males compared to controls; in contrast, Pdyn expression was significantly lower in the MBH of GABAGRKO females and Kiss1 expression was unchanged compared to controls. Agrp expression was lower in the MBH of both GABAGRKO male and female mice compared to controls. No differences were observed in Tac2 and Pomc expression. Data sets are mean ± SEM. Statistical significance was considered at p &amp;lt; 0.05. Conclusions: The deletion of GR in GABAergic neurons results in early puberty onset, sexually dimorphic changes in Pdyn mRNA levels and downregulation of Agrp mRNA expression in male and female GABAGRKO mice. These findings suggest a role of glucocorticoids acting on GR in GABAergic neurons upstream of KNDy neurons to restrain the timing of their activation to initiate puberty. Presentation: Thursday, June 15, 2023

Reproductive toxicity following in utero and lactational exposure to a human-relevant phthalate mixture in rats.

This rodent (Wistar rats) study examined reproductive effects of in utero/lactational exposure to a mixture of 6 antiandrogenic phthalates (PMix): diisobutyl phthalate, di-n-butyl phthalate, diisopentyl phthalate, butylbenzyl phthalate, di-2-ethylhexyl phthalate, and diisononyl phthalate. The PMix was defined based on exposure data from pregnant women in Brazil. Experimental groups were established by extrapolating the estimated human dose to rats (0.1 mg/kg/day), followed by up to 3 additional doses corresponding to 5, 1000, and 5000 times the starting rat dose: 0 (control), 0.1, 0.5, 100, and 500 mg/kg/day. The fetal experiment assessed gestational exposure effects on fetal gonads, whereas the postnatal experiment evaluated reproductive parameters in males and females after in utero and lactational exposure. Prenatal exposure decreased fetal testicular testosterone production at 0.5 and 500 mg/kg/day. PMix 500 also reduced mRNA expression of steroidogenesis-related genes, upregulated transcript expression of the retinoic acid-degrading enzyme Cyp26b1, and increased multinucleated gonocytes incidence in fetal testes. Postnatal assessment revealed antiandrogenic effects at the highest dose, including reduced anogenital distance, nipple retention, and decreased weight of reproductive organs. Early puberty onset (preputial separation) was observed at the lowest dose in males. In contrast, females did not show significant changes in fetal and adult endpoints. Overall, the PMix recapitulated early and late male rat phthalate syndrome phenotypes at the highest dose, but also induced some subtle changes at lower doses, which warrant confirmation and mechanistic assessments. Our data support the use of epidemiologically defined mixtures for exposure risk assessments over traditional toxicological approaches.

Insights from the maternal lineage of the F2 generation after exposure to an environmentally relevant dose of benzo(a)pyrene in the male rats of F0 generation.

Benzo(a)pyrene (BaP) is a substance with the potential to induce endocrine disruption in the F0 generation and cause adverse multigenerational effects (F1 generation) for reproductive parameters in rats. The objective of this study was to investigate the occurrence of transgenerational inheritance in the reproductive aspects of male and female rats belonging to the F2 generation (MF2). This investigation was conducted following the exposure of male rats from the F0 generation to BaP to assess potential effects on subsequent generation from the maternal lineage (F1). For that, juvenile male Wistar rats (F0) were orally exposed to BaP (0.1µg/kg/day) for 31 consecutive days. In adulthood, they were mated with untreated females to obtain female offspring (F1), which later produced the MF2. In the MF2 generation, both males and females exhibited increased body weight on postnatal day (PND) 1. In MF2 males, we observed delayed preputial separation, altered pup weight, reduced levels of follicle-stimulating hormone (FSH), increased intratesticular testosterone levels, decreased type A sperm, epididymal disturbances, reduced 5 α-reductase activity, increased testicular proliferation, and alterations in testicular antioxidant enzymes. In MF2 females, we noted morphological uterine enlargement, reduced sexual activity, and decreased progesterone levels. The findings suggest that the alterations observed in both MF2 males and females can be attributed to modifications in the sperm from F0 generation, which were subsequently transmitted to F1 females and MF2 generation due to BaP exposure.

Early-life exposure to acephate inhibits sexual development and induces testicular and ovarian toxicity in mice

Acephate is an organophosphate insecticide that exerts its toxicity by acting on the nervous system of insects. In addition to its action on the mammalian nervous system, acephate can also induce endocrine disruption of the reproductive system in mammals. However, the effects of acephate on sexual maturation and ovary development remain unclear. This study evaluated whether early-life exposure to acephate negatively impacts the male and female sexual maturation process and mature reproductive tissues. C57BL/6N mice were exposed to acephate (0, 0.3, 300 ppm) in drinking water from embryonic day 11.5 to ablactation, when the pups were four weeks old. Both sexes in the high-dose group experienced an early postnatal growth deficit, while females in the low-dose group continued to gain weight until 10 weeks of age. Exposure to acephate altered the anogenital index in females. Furthermore, preputial separation and vaginal opening were delayed in the high-dose group. At maturity, the weight of the seminal vesicles was decreased in the high-dose group. All treated groups exhibited increased vacuolation, accumulation of residual bodies, and degeneration in the testes. Furthermore, follicle regression was observed, and the healthy follicle number at each developmental stage was decreased in all treated groups. These results are probably due to the inhibition of the regulation by the hypothalamic-pituitary-gonadal axis and direct toxicity to reproductive organs. In conclusion, our study demonstrates that early-life exposure to acephate in mice may disrupt normal postnatal development, postpone puberty onset, and adversely affect reproductive functions during the reproductive period in both sexes.

Evaluation of The Effects of Carob (Ceratonia Siliqua L.) Fruits on the Puberty of Rats.

This study was planned to determine the effects of carob use on puberty because of the observation of early puberty or pubertal variants due to the long-term use of carob in our clinic. Forty-eight Wistar albino rats, on postnatal day 21, were assigned into two groups female (n=24) and male (n=24). Groups were divided into four groups Control, Carob-150, Carob-300, and Carob-600. Ceratonia Siliqua L. extract was given to rats in a 0.5% carboxymethylcellulose(CMC) solution. Carboxymethylcellulose (0.5 %) was given to the Control, Ceratonia Siliqua L. extract was given 150 mg/kg/day to the Carob-150, 300 mg/kg/day to the Carob-300, 600 mg/kg/day to the Carob-600 by oral gavage. The treatments were performed once daily until the first sign of puberty. Serum FSH, LH, Estradiol, Total Testosterone, Leptin, Glutathione, Glutathione peroxidase, and Malondialdehyde were studied with rat kits by the ELISA method. Testis, uterus and ovary tissue were examined histologically. The median time of preputial separation in male rats were 38th-31st-31st-31st days, Control, Carob-150, Carob-300, and Carob-600 (p=0.004). The median time of the vaginal opening day in female rats were 39th-31st-34th-31st days in the Control, Carob-150, Carob-300, and Carob-600 (p=0.059). FSH-LH-Testosterone(male)-Estradiol(female)-Leptin levels of groups are similar (p>0.05). Glutathione peroxidase levels were higher in the groups given C. Siliqua extract compared to the Control in both male (p=0.001) and female groups (p=0.008). Testicular and ovarian tissues were concordant with the pubertal period in all groups. As the dose of CS increased, it induced spermatogenesis and spermiogenesis, causing abnormal changes such as ondulation in the basement membrane, capillary dilatation, and increased congestion in male. Edema in the medulla gradually increased with increased dosage, and granulosa cell connections were separated in Carob-300 and Carob-600 in female. Our study demonstrated that C. siliqua caused early puberty and increased spermiogenesis and folliculogenesis. We discovered that antioxidant mechanisms might affect this. Further, it may cause tissue damage at high doses.

Effect of Different Doses of Volatile Oil Extract of Rosemary in Suckling Rats on Some Physiological Parameters and Pups

Background: The current study was carried out in the animal house-College of the Veterinary Medicine/University of Mosul-Iraq. The study aimed to evaluate the effects of volatile oil extract of rosemary on some physiological and reproductive parameters of suckling female rats and their pups. Methods: 24 suckling female rats were used and have been distributed randomly to four groups 6 suckling female rats for each group. The first group was considered a control group that was administered only with corn oil orally while the other groups were treated orally with volatile oil extract of rosemary 250,500, 1000 mg/kg body weight for 21 days (time of suckling). Result: Results revealed that the treatment of suckling female rats with volatile oil extract of rosemary caused a significant P less than 0.05 decrease in body weight of suckling female and its pups in 7,14,21 days of suckling time and an increase significantly P less than 0.05 the mortality of suckling female and its pups, as well as increase the growth factor of pups(days) significantly which include (open of ears, obtained of hair, obtain of teeth and opened of the eyes), compared with the control group. Doses of volatile oils extract of rosemary (250, 500, 1000 mg/kg b.w.) caused a significant P less than 0.05 increase of testicular descent and primary preputial separation of male suckling pups (days) and a decrease significantly P less than 0.05 Ano-gential distance (AGD)/mm, as well as increased vaginal opening significantly P less than 0.05 of female suckling pups compared with the control group. There was a significant decrease P less than 0.05 in each of estrogen, prolactin, FSH and LH in all doses of rosemary oil used in the study, while there was no significant difference in progesterone copared to the control group in suckling females.

Chronic periadolescent leuprolide exposure affects the development of reproductive physiology and behavior of female and male rats differently, but both mature after treatment termination

BackgroundGnRH agonists have been used to halt the development of puberty in children with precocious puberty since the 1980s. Recently, drugs like Lupron Depot® (leuprolide acetate), have been used to suppress pubertal progression in adolescents who are questioning their gender identity. However, few preclinical studies have been conducted to investigate potential effects of using GnRH agonists in this context.MethodsThe present study tested the effects of daily leuprolide treatment (50 µg/kg, postnatal day (PD) 25–50) on pubertal onset in female (i.e., vaginal opening) and male (i.e., preputial separation) Long-Evans rats. The first estrous cycle immediately after vaginal opening was also measured. Sexual behavior and sexual motivation were tested using the partner-preference paradigm. Female rats were tested during the first behavioral estrus after treatment ended (between PD 51–64). Male rats were tested weekly for four consecutive weeks starting three days after treatment ended (PD 53).ResultsConsistent with previous findings, leuprolide significantly delayed pubertal onset in both female and male rats. In addition, the first estrous cycle during the treatment period was disrupted by leuprolide, as indicated by a failure to cycle into estrus after vaginal opening until treatment ended. However, leuprolide affected neither sexual motivation nor fertility when female rats were tested within 14 days of leuprolide treatment ending. In contrast, the development of copulatory behavior and sexual motivation was significantly delayed by leuprolide in male rats; however, mature reproductive behavior was observed by the fourth week post-treatment.ConclusionsTaken together with previous findings, the present results indicate that male rats may be more sensitive to periadolescent leuprolide administration, taking longer to overcome the effects of leuprolide than female rats. Nevertheless, not long after leuprolide treatment is discontinued, sex-typical reproductive physiology and behavior emerge fully in female and male rats, indicating that the drug’s effects are not permanent. If translatable to humans, leuprolide may be a reversible option to give adolescents more time to consider their gender identity with minimal long-term effects on sexual development.

Safety evaluation of ondansetron after gestational exposure on male reproductive parameters in rats

Ondansetron is a 5HT3 receptor antagonist widely used to treat hyperemesis gravidarum, although its safety is still questionable. Since 5HT3 receptors, which are the target of this drug, can interfere with brain development through changes in neurotransmitter levels, this study evaluated whether the prenatal exposure to this drug could compromise reproductive and behavioral parameters in male offspring. Pregnant rats were treated with ondansetron (1.7 and 2.5 mg/kg/body weight; gavage), from gestational day 1–21. No exposure-related changes in clinical signs, body weight, food consumption, pregnancy length, and necropsy findings were observed in dams. Ondansetron exposure did not alter the anogenital distance or age of preputial separation in male offspring. Similarly, males exposed to therapeutic doses of ondansetron did not exhibit changes in play behavior. In adulthood, there were no changes in sperm parameters, as well as in testosterone level, sexual behavior and fertility. Furthermore, ondansetron did not interfere with testicular and epididymal histology, and with androgen receptor expression in hypothalamus. In conclusion, prenatal exposure to ondansetron did not cause maternal toxicity, as well as did not interfere with reproductive parameters of male offspring, indicating its safety after gestational exposure in rats.

ODP405 Effects of Gonadotrope-Specific Knockout of Creb on HPG Axis Function in Mice

Abstract Background and Objective Reproduction is regulated by FSH and LH, which are synthesized and secreted by pituitary gonadotropes in response to pulsatile hypothalamic GnRH in a frequency dependent manner. Low and high GnRH pulse frequencies preferentially increase Fshb and Lhb expression, respectively, mediated by preferential coupling to Gαs-PKA-Creb and Gαq-PKC-MAPK-AP1 signaling cascades in vitro. The mouse Fshb proximal promoter contains a half-AP1/half-CRE recognition motif that binds Creb in vitro, and mutation of this half-CRE results in loss of preferential GnRH stimulation of Fshb at low pulse frequencies in immortalized gonadotrope LβT2 cells. Therefore, the purpose of this study was to identify the direct role of Creb on gonadotropin regulation in vivo. We hypothesized that gonadotrope-specific deletion of Creb would influence pubertal timing and/or fertility due to reduced Fshb expression. Methods and Results Gonadotrope-specific Creb knockout (Creb1 fx/fx / Gnrhr GRIC/+; Creb KO) and control (Creb1 fx/fx) mice were generated by Cre-Lox recombination and markers for pubertal onset were evaluated. We observed no differences in body weights of Creb KO and littermate control mice in either females or males from postnatal day (PND) 21 to 3 months of age (n=9/genotype/sex). The age of vaginal opening, a marker of puberty onset in female mice, was not significantly different between female Creb KO (PND 28.4±0.5; n=21) and littermate control (PND 29.5±0.6; n=17) mice. Similarly, the age of first estrus, determined by vaginal lavage and cytology, was not significantly different between Creb KO mice (PND 37.6±1.9; n=9) and littermate controls (PND 36.6±2.2; n=8). Estrous cyclicity was normal over a three-week period in Creb KO and control mice, which spent similar amounts of time in diestrus/metestrus, proestrus, or estrus. Male Creb KO mice tended to show preputial separation at a younger age (PND 28.8±0.3; n=19) compared to littermate control (PND 29.6±0.3; n=22) mice, but the difference did not reach statistical significance (p=0. 06). Conclusions Deletion of Creb from pituitary gonadotropes did not affect the timing of pubertal onset in female mice, but we did observe a trend for male Creb KO mice to have advanced puberty, as measured by preputial separation. Normal pubertal timing does not preclude potential effects of Creb-mediated gonadotropin regulation on folliculogenesis, spermatogenesis, or fertility (including litter size), for which studies are ongoing. Presentation: No date and time listed

Reproductive and developmental toxicology studies of iminosugar UV-4

UV-4 (N‐(9‐methoxynonyl)‐1‐deoxynojirimycin) is a host-targeted antiviral agent, which targets mammalian proteins (endoplasmic reticulum glucosidases) rather than virally encoded proteins. This mechanism confers both broad-spectrum activity and low potential for generation of viral drug resistance mutations. Reproductive and developmental studies of UV-4 evaluated effects on fertility and early embryonic development in rats, embryo-fetal development in rats and rabbits, and pre- and postnatal development including maternal function in rats. All reproductive and developmental studies conducted achieved dose levels where parental toxicity (generally decreased body weight, decreased food consumption and adverse clinical signs) were observed. Toxicokinetic evaluations confirmed UV-4 crossed the placenta exposing fetal rats and rabbits in utero. Adverse findings in reproductive and developmental studies included decreases in sperm motility with histopathology correlates, visceral and skeletal malformations, changes in eye opening, air drop reflex, vaginal opening and preputial separation. The combined results of the fertility and early embryonic developmental study and pre- and postnatal study suggested that there may be an increased risk for male fertility. These effects are similar to those reported in pre-clinical studies of the structurally related drug Miglustat (N-butyl-1-deoxynojirimycin), therefore UV-4 may have risk of developmental or reproductive adverse outcomes in humans similar to existing approved agents in this drug class.

Reproductive toxicity of maternal exposure to di(2-ethylhexyl)phthalate and butyl paraben (alone or in association) on both male and female Wistar offspring.

Parabens and phthalates are commonly found as contaminants in human fluids and are able to provoke reproductive toxicity, being considered endocrine disruptors. To evaluate the effects of phthalate and paraben, alone or in combination, on reproductive development of the offspring, female pregnant Wistar rats were allocated in six experimental groups: Three control groups (gavage [CG], subcutaneous [CS], and gavage + subcutaneous) received corn oil as vehicle, and the remaining groups were exposed to di(2-ethylhexyl)phthalate (DEHP) (500 mg/kg, gavage), butyl paraben (BP) (100 mg/kg, subcutaneously), or MIX (DEHP + BP), from Gestational Day 12 until Postnatal Day (PND) 21. The following parameters were assessed on the offspring: anogenital distance and weight at PND 1, nipple counting at PND 13, puberty onset, estrous cycle, weights of reproductive and detoxifying organs, histological evaluation of reproductive organs, and sperm evaluations (counts, morphology, and motility). Female pups from MIX group presented reduced body weight at PND 1, lower AGD, and decreased endometrium thickness. Male animals showed decreased body weight at PND 1 and lower number of Sertoli cells on DEHP and MIX groups, MIX group revealed increase of abnormal seminiferous tubules, DEHP animals presented delayed preputial separation and higher percentage of immotile sperms, and BP males presented diminished number of Leydig cells. In conclusion, the male offspring was more susceptible to DEHP toxicity; even when mixed to paraben, the main negative effects observed seem to be due to antiandrogenic phthalate action. On the other hand, DEHP seems to be necessary to improve the effects of BP on reducing estrogen-dependent and increasing androgen-dependent events.