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| Status |
Public on Jun 21, 2023 |
| Title |
Sterilization of female rats by single-injection of estrogen: proof-of-concept for a nonsurgical alternative |
| Organism |
Rattus norvegicus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Reproductive sterilization via surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive diseases. The present study explored the feasibility of using a single-injection method for inducing sterility in female animals as an alternative to surgical sterilization. The idea was based upon our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin, the neuropeptide that triggers and regulates pulsatile secretion of GnRH in the hypothalamus. In this study, neonatal female rats were dosed with estradiol benzoate (EB) by daily injection for 11 days or subcutaneously implanted with an EB-containing Silastic capsule designed to release EB over a 2- to 3-week period. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer Kisspeptin neurons in their hypothalami, but their GnRH-LH axis retained responsiveness to Kisspeptin stimulation. To simplify usage, an injectable EB carrier was developed in the forms of biodegradable microspheres or pellets with pharmacokinetics comparable to the EB-containing Silastic capsule. A single injection of EB microspheres (300 µg EB) to neonatal rats made them sterile and female beagles implanted with an EB pellet at a neonatal age had lower KISS expression in their hypothalami. No concerning health impact other than infertility was observed in animals that received EB treatment at a neonatal age. Taken together, this study shows that neonatal EB administration could be developed as a non-surgical animal sterilization method.
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| Overall design |
Sprague Dawley rats used in this study were purchased from Charles River Laboratory and bred at the University of Illinois Division of Animal Resources and maintained under controlled lighting (12 h light/12 h dark) with continuous access to food and water. Rats were acclimatized for one week prior to beginning the experimental procedures. Breeder pairs were housed together for 2 weeks; pregnant females were housed individually, and pups were weaned at 21 days of age. Female neonates were treated with Silastic capsules containing the vehicle sesame oil implanted on postnatal day (PND) 0.5 (SC300). To investigate differential gene expression in individual cells of the hypothalamus, tissues were collected from 3 intact control and 3 SC300 rats, and pooled by group. For single-cell RNA sequencing (scRNAseq) analyses, hypothalamic tissues that contain AVPV and ARC area were cut into small pieces, dissociated into single-cells, and converted into individually barcoded cDNA libraries with the Single-Cell 3’ Chromium kit version 3 from 10X Genomics (Pleasanton, CA), following the manufacturer’s protocols. The 10X Chromium instrument separates thousands of single cells into Gel Bead Emulsions (GEMs) that add a barcode to the mRNA from each individual cell. Following ds-cDNA synthesis, individually barcoded libraries compatible with the Illumina chemistry were constructed. The final libraries were quantitated on Qubit and the average size determined on the AATI Fragment Analyzer (Advanced Analytics, Ames, IA). Libraries were pooled evenly, and the final pool was diluted to 5 nM concentration and further quantitated by qPCR on a Bio-Rad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA). The final library pool was sequenced on a 2x150nt S4 lane in a NovaSeq 6000. Basecalling and demultiplexing of raw data was done with the mkfastq command of the software Cell Ranger 3.0.2 (10x Genomics). Single-cell expression was initially analyzed to perform quality control, sample de-multiplexing, barcode processing, and single-cell 3′ gene counting. Sequencing reads were aligned to the Ensembl96 transcriptome using the Cell Ranger suite with default parameters. Samples were merged using Cellranger aggregate function with default parameters. A total of 4,488 and 4,524 cells from hypothalami of Control and SC300 rats, respectively, was analyzed. Mean raw reads per cell were 151,484 and 147,555 in each sample. Median numbers of expressed genes per cell were 2,479 and 2,530 in each sample. Further analysis was performed in R (v3.5) using the cellrangerRkit (v2.2.0), Seurat package (v3.0.0), and Monocle package (v2.8.0).
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| Contributor(s) |
Park C, Minabe S, Hess R, Lin P, Zhou S, Bashir ST, Barakat R, Gal A, Ko CJ |
| Citation(s) |
37316510 |
| Submission date |
Dec 14, 2022 |
| Last update date |
Jun 23, 2023 |
| Contact name |
CheMyong Jay Ko |
| E-mail(s) |
jayko@illinois.edu
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| Organization name |
University of Illinois at Urbana Champaign
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| Department |
Comparative Biosciences
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| Street address |
2001 S. Lincoln Ave.
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| City |
Urbana |
| State/province |
Illinois |
| ZIP/Postal code |
61802 |
| Country |
USA |
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| Platforms (1) |
| GPL25947 |
Illumina NovaSeq 6000 (Rattus norvegicus) |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA912242 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE221002_RAW.tar |
86.4 Mb |
(http)(custom) |
TAR (of MTX, TSV) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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