Sterilization of female rats by single-injection of estrogen: proof-of-concept for a nonsurgical alternative. Sterilization of female rats by single-injection of estrogen: proof-of-concept for a nonsurgical alternative

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. 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).

通过手术摘除性腺的生殖绝育手段被广泛提倡，用于管控动物种群数量（尤其是家养宠物），并预防生殖系统疾病。本研究探讨了采用单次注射法诱导雌性动物不育的可行性，以此作为手术绝育的替代方案。该思路基于我们近期的一项发现：向新生大鼠每日重复注射雌激素，可破坏下丘脑内Kisspeptin（吻肽）的表达——该神经肽可触发并调节下丘脑内促性腺激素释放激素（Gonadotropin-Releasing Hormone, GnRH）的脉冲分泌。

本研究中，新生雌性大鼠通过每日注射苯甲酸雌二醇（Estradiol Benzoate, EB）持续11天，或皮下植入可在2~3周内释放EB的Silastic胶囊进行处理。两种处理方式的大鼠均未表现出发情周期，无排卵且不育。经EB处理的大鼠下丘脑内Kisspeptin神经元数量更少，但它们的GnRH-促黄体生成素（Luteinizing Hormone, LH）轴仍可对Kisspeptin刺激产生响应。

为简化使用方式，我们开发了可注射型EB载体，采用生物可降解微球或微丸剂型，其药代动力学与含EB的Silastic胶囊相当。向新生大鼠单次注射EB微球（300 μg EB）即可使其不育；新生时期植入EB微丸的雌性比格犬，其下丘脑内KISS基因表达水平更低。在新生期接受EB处理的动物中，除不育外未观察到其他显著健康影响。

综上，本研究表明新生期给予EB可开发为一种非手术式动物绝育方法。

实验总体设计：本研究使用的斯普拉格-道利（Sprague Dawley, SD）大鼠购自Charles River Laboratory，在伊利诺伊大学动物资源部繁育饲养，饲养环境光照可控（12小时光照/12小时黑暗），动物可自由采食饮水。实验操作开始前，大鼠需适应性饲养1周。种鼠配对合笼2周；孕鼠单独饲养，幼崽于21日龄时断奶。新生雌性大鼠于出生后第0.5天（PND 0.5）植入含有空白溶剂芝麻油的Silastic胶囊（SC300组）。

为研究下丘脑单个细胞的差异基因表达，我们分别从3只正常对照大鼠和3只SC300组大鼠体内采集下丘脑组织，按组混合。对于单细胞RNA测序（single-cell RNA sequencing, scRNAseq）分析，将包含AVPV和ARC区域的下丘脑组织剪碎，解离为单细胞悬液，随后按照10X Genomics（美国加州普莱森顿）的Single-Cell 3’ Chromium试剂盒v3版的厂商说明书，构建带有单个细胞条形码的cDNA文库。10X Chromium仪器可将数千个单细胞分离至凝胶珠微滴（Gel Bead Emulsions, GEMs）中，为每个单细胞的mRNA添加独特条形码。完成双链cDNA合成后，构建适配Illumina测序化学的带条形码文库。最终文库通过Qubit进行定量，并通过AATI Fragment Analyzer（美国爱荷华州埃姆斯Advanced Analytics公司）测定平均片段大小。将文库按等量混合，将终混合液稀释至5 nM浓度，随后通过Bio-Rad CFX Connect实时定量PCR系统（美国加州Bio-Rad实验室有限公司）进行定量。最终文库混合液在NovaSeq 6000的2x150nt S4测序流槽上完成测序。原始数据的碱基识别与拆分使用10X Genomics的Cell Ranger 3.0.2软件的mkfastq命令完成。

初始单细胞表达分析用于完成质量控制、样本拆分、条形码处理及单细胞3’基因计数。测序读数通过Cell Ranger套件以默认参数比对至Ensembl96转录组。使用Cellranger aggregate函数的默认参数合并样本。本研究共分析了对照组和SC300组大鼠下丘脑的4488个和4524个细胞。每组样本的平均原始reads数分别为151484和147555；每组样本的每个细胞表达基因数中位数分别为2479和2530。后续分析使用R语言（v3.5），借助cellrangerRkit（v2.2.0）、Seurat包（v3.0.0）及Monocle包（v2.8.0）完成。