Progress 09/02/16 to 08/31/21
Outputs
Target Audience:Target audience include graduate and undergraduate students who participate in this research in laboratory setting, and other scientists who works endocrine-disrupting chemicals (EDCs) especially phthalates and their effects on female reproductive and endocrine system. In addition, it is public that are likely to be exposed to EDCs, such as phthalates that are likely to learn from the results of the study. The regulatory agencies also may consider the findings of this project in their future regulatory activities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Several undergraduate and graduate students have been trained in various in vivo and in vitro procedures as related to study effects of EDCs on female reproductive functions, including dosing, monitoring various reproductive parameters, such as monitoring of pubertal age,regularity of estrous cycles, pregnancy rate, and litter size, tissue collection and processing for histology, and follicle classification and counting. In addition, studentsalso learned data collection, analysis, and presentation. How have the results been disseminated to communities of interest?Several undergraduate students presented their work in undergraduate student symposia as slide or poster presentations and as honor thesis presentations. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
An impact statement: Endocrine-disrupting chemicals (EDCs)are common in the environment. Public are continuously exposed to these chemicals in daily bases through food, packaging, household items, personal care products and even health care products. Phthalates are one such class of chemicals in the environment. Almost 100% of the US population has detectable levels of phthalates in their urine or blood. The basic research with lab animals suggested that the phthalates have clear antiandrogenic effects, especially if exposure occurs during development, which is supported with some epidemiological data. Similarly, high dose phthalates also had effects on adult females and ovary. However, more recent data have shown that developmental exposures to even lower dose to phthalates could induce female fertility problems. Our data clearly confirmed the antiandrogenic effects in male rats. However, the effects on female fertility and ovary were not so clear-cut based on the parameters that we measured. Project activities and accomplishment: We had two objectives in this project: 1-The first objective was to investigate how the phthalates affect the number and composition of the follicles in the ovary. Follicles are the functional units of the ovary and are important to have normal numbers and compostion for a normal female reproductive function and health. In addition, as part of this objective, we investigated the female reproductive parameters to assess female fertility. As for the ovarian follicle count, there were no effects of DEHP and DINP on the follicle number and compassion at 2 months of age. It is possible that there could be effects when the females are older. In addition, we examined female reproductive parameters, including pubertal age as determined by vaginal opening, regularity of cycle followed monthly between 2 and 18 months of age, pregnancy rate and litter size at 2, 6 12, and 18 months of age. We also examined the ovarian and uterine weight at 2, 6 12 and 18 months of age. The results showed that the regularity of cycles were affected with 240 mg DEHP only at 7 month of age (p < 0.05, with t-test), but the cycles were not affected in any other month or with any other doses of DEHP or DINP. In addition, thelitter size was reduced by 500 mg DINP only at 12 month of age (p < 0.05, with t- test).The litter size wasnot affectedin any other times, or with any other doses of DNIP or DEHP. No significant effects on ovarian or uterine weights. 2-The second objective to project was to examine the effects of phthalates on global gene expression using RNA-seq technology. There were no significant effects of phthalates at 2, 6 and 12 months. In summary, the results suggest the effects of the phthalates on female reproduction and the ovary in ratswere minimal despite relatively high dose that we used.Considering there are reports with lower dose of phthalates on femalereproductive functionor ovary in mice, and the lack of effects in the rats in our project, it is possible thatthe effects of phthalates arespecies-specific, mouse being more sensitive to phthalates. Species-specific effects of phthalates may require a closer examination.
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Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Target audience include undergraduate students who participate into this research in laboratory setting or vituallyonline, and other scientists who work in the area of endocrine disrupting chemicals especially phthalates and their effects on female reproductive and endocrine system. Changes/Problems:The COVID-19 pandemic slow down the progress in follicle counting since students are not allowed to campus in Spring and Summer semester, and only allowed limited basis in Fall. However, students are more likely return in the lab during Spring, and we will continue with follicle counting in the samples from the second batch. What opportunities for training and professional development has the project provided?Sneha Manikandan and Krista Collins, who were graduated in May 2020 majoring in Cell Biology and Neuroscience and Psychology respectively, Madeline Procopi and JuliaSzymanskia senior and junior majoring in Animal Sciences, respectively were trained in various procedures, including tissue collection, histology cassette preparation, paraffin embedding, section selection, quality control of unstained and stained sections, follicle classification and counting. How have the results been disseminated to communities of interest?Sneha Manikandan and Krista Collins presented their work for honor thesis to their thesis committee in April of 2019. JuliaSzymanskipresented her work at Rutgers' Project SUPER Poster presentation recently. What do you plan to do during the next reporting period to accomplish the goals?We plan to perform follicle counting the second batch of animal with additional females whose ovaries were similarly collected at PND 60 postnatal day 60 females and combine the data with the current set and reanalyze it.
Impacts
What was accomplished under these goals?
The current study examines the effect of phthalateson the ovary, the female gonad. The phthalates areplasticizersthat commonly exist in our daily environment as part ofarray of consumer goods, medical supplies/equipment, and building materials, as well personal care products and medications. Their effects on male and testis are known, but potential effects in females is less known. This study aims to fill this gap. To this end, during this period, we have assessed follicle numbers in F1 females followingtreatment of timed-pregnant female rats via oral gavage with corn oil (Control) and 24, 240, and 500 mg/kg/day of DEHP or DINP between embryonic day 11 to postnatal (PND) 7. Ovaries were collected on PND 60, and were formalin-fixed, paraffin-embedded, andsectioned at 5 µm. Follicles were counted in H-E stained sections (total of 6 sections in the middle of ovary 100 µm apart). The follicles wereclassified into primordial, primary, secondary,preantral, early antral, and mid-to-late antral follicles according to previously published protocols [1, 2].In addition, corpora lutea (CL) and atretic follicles were counted. We had 3-4 females from different litters in each treatment groups.The counting was performed blindly by two individuals and data was averaged.This was the first batch of two-batch experiment. The results were analyzed using Graph Pad Prism using one-way ANOVA followed byDunnett's multiple comparison and alpha was set at 0.05. While we originally planned to count sections from the second batch animals during spring and summer of 2021, but due to the COVID-19 restrictions, the count was delayed. Based on the currently available follicle count results, the effects of the DEHP and DINP treatment on the follicle and CL numbers and follicle composition were not significant. However, there was a trend for a reduction (p < 0.1) in primordial and early antral follicles in group treated with 500 mg/kg/day DEHP and in primary follicles in group treated with 24 mg/kg/day DEHP. The lack of a statistically significant effect may be due to a relatively small sample number. To incrase the sample size, thefollicle count and classification will be performed with sections from additional 4-5 females. Once those sections were counted, the data will be combined from both batches. We believe that this will would give a clearer picture for the effects phthalates on the ovarian folliculogenesis. 1.Hannon, P.R., et al.,Di(2-ethylhexyl) phthalate inhibits antral follicle growth, induces atresia, and inhibits steroid hormone production in cultured mouse antral follicles.Toxicol Appl Pharmacol, 2015.284(1): p. 42-53. 2.Armenti, A.E., et al.,Developmental methoxychlor exposure affects multiple reproductive parameters and ovarian folliculogenesis and gene expression in adult rats.Toxicol Appl Pharmacol, 2008.233(2): p. 286-96.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Target audience include undergraduate students who participate in this research in laboratory setting, and other scientists who work in the area of endocrine disrupting chemicals especially phthalates and their effects on female reproductive and endocrine system. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Rutgers University undergraduate students, Sneha Manikandan (senior, majoring in Cell Biology and Neuroscience), Krista Collins (senior, majoring in Psychology) and Madeline Procopi (junior, majoring in Animal Sciences) have been trained in various procedures, including tissue collection, histology cassette preparation, paraffin embedding, section selection, quality control of unstained and stained sections, follicle classification and counting. Joseph Yunga Tigre, who graduated from Rutgers with an undergraduate degree in Biological Sciences in May 2019 was also trained in including tissue collection, histology cassette preparation, paraffin embedding. How have the results been disseminated to communities of interest?Joseph Yunga Tigrepresented his work for honor thesis to his thesis committee. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue with studying the effect of DEHP and DINP on folliculogenesis by completing the follicle counts for PND 60 ovaries and correlating the finding with gene expression studies that is also on-going.
Impacts
What was accomplished under these goals?
During this period, we have done some preparatory work to determine the effectsof DEHP and DINP on folliculogenesis, which is part of the Objective 1, namely"to investigate when and how developmental exposures to DEHP and DINP alter ovarian follicular dynamics and female fertility".There has been evidence supporting the effects of phthalates on folliculogenesis by other labs[1]. To further our understanding in this area, especially with DINP, we wanted to study the effects on follicular dynamics. To this end, we collected one of the ovaries from females rats that were developmentally exposed to various doses of DINP and DEHP. Ovaries were collected at postnatal day (PND) 60, 180 and 360. The contralateral ovaries were used for gene expression analysis. During this reporting period, we focused on the follicular count in the ovaries collected at PND 60. Formaldehyde-fixed, paraffin-embedded tissues were serially sectioned at 5 µm. Approximately 250 sections were collected starting from one end of the ovaries and passing the largest cross-sectional areas of the ovary at last 100 µm. Since counting 250 slides not feasible, we selected two neighboring sections from the largest cross-sectional area approximately at middle of the ovary. Four additional sections, two neighboring sections at 100 µm upstream and two neighboring sections at 100 µm downstream from the middle were also selected. These sections were stained with hematoxylin-eosin (HE) staining for follicle counting. This selection approach was previously used to estimate the changes in the follicular composition in rat ovaries[2]. The H-E staining was completed.The follicle counting is currently in progress.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Target audience includeundergraduate students who participate in this research in laboratory setting, andother scientists who work in the area of endocrine disrupting chemicals especially phthlates and their effects on female reproductive and endocrine system. Changes/Problems:We have added a new objective for the project: In addition to studying effects of phthalates (i.e., DiNP and DEHP) on the female reproduction, we decided to study their effects on the glucose metabolism and expression of relevant genes in tissues, such as liver, pancreas, and adipose tissues. We decided to look at these effects because, as mentioned above, phthalates act through PPAR-signaling [1-3], which is known to affect glucose homeostasis [4] and adipogenesis [5]. In addition, human epidemiological studies support the effects of phthalates on glucose metabolism [6]. Moreover, the effects in males are well-known, but information in females are limited. Thus, there is an information gap. Therefore, we added a third objective to our project, which is: To study the developmental effects of DiNP and DEHP on glucose metabolism and on expression of genes relevant to glucose metabolism in liver, pancreas, and fat tissues. To this end, we (i) use oral glucose tolerance test (OGTT), insulin tolerance test (ITT) throughout the lifespan of the animals, (ii) monitor organ weights for liver and various visceral fat tissues (perimetrial, perirenal, and abdominal) and (iii) measure expression of genes in liver, abdominal fat, and pancreas using qRT-PCR. This change is already approved by Rutgers' IACUC. What opportunities for training and professional development has the project provided?Undergraduate students have been trained in various procedures, including oral glucose tolerance test and insulin tolerance test, and tissue collection, as well as data collection, analysis, and presentation. How have the results been disseminated to communities of interest?Undergraduate student BryceJurkouich (Stockton University, NJ), who worked as a participant of NIH-funded Summer Undergraduate Research Fellowship (SURF) program, presented a short (6-min) power point presentation to his peers, program organizers, and participating Rutgers faculty hosts at the end of his 10-week training. In addition, Madeline Procopi (Animal Science major), who join my lab as Aresty Summer Undergraduate Student researcher presented a poster at the end of her 10-week program. What do you plan to do during the next reporting period to accomplish the goals?It is worth to note that the effects that we reported above are relatively modest. The following may have contributed to the lack of more robust effects: (i) small sample size, (ii) animals were too young to have the full effects of treatments, and/or (iii) variability of the data due to varying reproductive cycle stages when we conducted OGTT and ITT. All of these potential reasons will be addressed in the future by increasing sample size, examining the effects in aged-females (12-month-old), and taking the cycle stage into consideration for all data (e.g., OGTT, and ITT) as well as tissue collections. In addition, it is also worth to note that while DEHP caused insulin resistance, DINP adversely affected the glucose clearance. Therefore, we will examine the potential mechanisms of these differential effects as well as any effect on adipogenesis by studying expression of relevant genes in liver, pancreas, and adipose tissue.
Impacts
What was accomplished under these goals?
There is an increasing prevalence of obesity and metabolic disorders, such as type II diabetes among human populations throughout the world. While factors, such as malnutrition or sedentary life style can play roles in this problem, endocrine disrupting chemicals (EDCs) can also contribute. In addition, EDCs, such as phthalates act through PPAR-signaling, which is known to affect glucose homeostasis and adipogenesis. Thus, it is important to study the developmental effects of phthalates on glucose metabolism and on the expression of genes that can play role in glucose homeostasis. Information obtained from our research can be used in prevention and treatment of metabolic disorders, induced by the developmental exposures to EDCs. During this research period, timed-pregnant Fischer CDF rats (F0; N=3-4) were orally treated with oil (Control) and 24, 240, and 500 mg di-iso-nonyl phthalate (DINP) or di(2-ethylhexyl) phthalate (DEHP) per kg BW per day between embryonic day (E) E11 and postnatal day (PND) 7 (sperm positive vaginal smear day= E0; day of birth of litter = PND0). In F1 females, blood glucose levels (mg/dL) were measured before and at 15, 30, 60, 90, and 120 min after oral glucose (2 mg/kg) gavage or after insulin (1.0 U/kg) injection for oral glucose tolerance test (OGTT) or insulin tolerance test (ITT), respectively, between PND130 and 140. OGTT was performed after a 16-hour fasting and ITT was performed after a 4-hour fasting and both values were expressed as area under curve (AUC). The highest DEHP (500 mg/kg/day) tended to increase (p = 0.09) AUC values for ITT, suggesting that developmental treatment with DEHP induces insulin resistance. In contrast, highest dose of DINP (500 mg/kg/day) significantly increased (p = 0.05) the AUC values for the OGTT, suggesting that developmental treatment adversely affect glucose clearance from the blood. In addition, since, insulin resistance is associated liver pathologies [8, 9] and adipogenesis [10, 11] in rats and humans, we examined the liver weight as well as weight of perimetrical, perirenal, and abdominal fat tissues. Highest doses DINP tended to increase (p = 0.06) the liver weight. However, fat accumulation was not significantly affected.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Target audience include graduate and undergraduate students who participate in this research in laboratory setting, andother scientists who works in the area of ovarian biology and related areas. Changes/Problems:Due to issues with full comsumption of thephthalates with vanilla wafers, oral gavage method will be used to expose the animals to phthalates. In addition, an additional higher dose (500 mg/kg)will be used for higher dose to ensure the effectiveness of the treatment and have at least a positive control in our treatment protocol. What opportunities for training and professional development has the project provided? Undergraduate and graduate students have been trained in various procedures, including in vivo procedures as related to study effects of EDCs on female reproductive functions, including dosing, tissue collection, monitoring pubertal age regularity of estrous cycle; and metabolic procedures such as oral glucose tolerance testand insulin tolerance test. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period,our plan is to repeat the study, and will add a higher dose (500 mg/kg/day) DiNP and DEHP to increase the likelihood of observing an effect. In addition, rather than giving the treatments via wafer, we will give themvia oral gavage, which we did not use in the first study to avoid the stress of handling the pregnant or lactating dams during oral application. However, since all animals would be similarly handled and exposed to same stress, we think this should not pose any problem. We will make this modification because, as described above, some animals were not fully consuming wafers that were laced with highest doses of DiNP and DEHP, which may have introduced additional variation to the experiment. In addition, in this modified study,we will conduct follicle count analysis and global gene expression analysis in the ovary, besides above described analyses.
Impacts
What was accomplished under these goals?
As proposed, timed-pregnant Fischer rats (n=4-5 dams, F0) were treated between embryonic day (E) 11 and postnatal day (PND) 7 with three doses of DiNP and DEHP (240 µg, 24 mg, or 240 mg/kg/day). The corn oil was used as control vehicle. The daily treatments were infused in vanilla wafers, which were placed in the cages. In preliminary studies, we tested vanilla wafers with all doses in adult female rats, which consumed all of their wafers with no issues. In addition, prior to the study, the dams were acclimatized with the wafer only for 2-3 days before the actual treatment started on E11. Thus, when we gave the wafers with treatments to the dams starting E11, all wafers were consumed by the dams in a few minutes after placing the wafers in animal's cages, except the highest dose (240 mg) of DiNP and DEHP. More specifically, although all 240 mg DiNP dams consumed all of their wafers during the initial few days of E treatment period, some of the dams either did not eat or partially ate the wafer at later stages of E period. Again, some dams did not eat their wafers during PND treatment period either. As for 240 mg DEHP, all dams consumed their wafers during E treatment period, except one dam which did not eat her wafer in last two days of E treatment period. However, all of the dams had issues consuming the wafers during entire PND period. To 'rescue' the experiment, any treatment amount that was not consumed via the wafer was estimated by the the remaining portion of the wafer andgiven to the dams orally with a micropipette. The following parameters were assessed in all treated F1 generation females and males for certain parameters [i.e., weekly body weights (BW) between PND1 and 28 and anogenital distance (AGD) on PND 1 and 6]: Weekly BW between PND 1 - 63 Monthly BW measurement at 4th, 5th and 6th month of age AGD on PND 1 and PND 6 both in males and females Pubertal age, the day of vaginal opening Regularity of reproductive cycle between the day of vaginal opening and 6th month Litter size Sex ratio In addition, as phthalates are known to affect metabolic parameters, we have assessed the effects of DEHP or DiNP treatments on metabolic parameters, specifically oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) at 6th month of age, even though these assays were not included in the original application. Results None of the treatments had significant effect on BW and organ weights. The treatment did not significantly alter any of the reproductive parameters listed above, namely AGD, pubertal age, regularity of estrous cycles, litter size, and sex ratio. In addition, none of the treatments had any effects on OGTT and ITT at 6 month of age. To asses if the treatment cause early reproductive aging, we are currently monitoring the regularity of estrous cycles between 6-9 and will assess the litter size at 9-10 month. In addition, we plan to collect ovarian tissue at 1 year of age and examine follicle number and global gene expression in the ovaries as proposed.
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Progress 09/02/16 to 09/30/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Due to short duration of project perod (Sept 02, 2016 - Sept 30, 2016), there is nothing to report at this reporting period.
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