Single neonatal estrogen implant sterilizes female animals by decreasing hypothalamic KISS1 expression

Abstract

Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2-3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

Figure 1

Experimental design. (A) Female rats on PND 0.5 were placed into one of the following treatment groups and followed for seven months: control (silicone capsule filled with oil); EBx11 (subcutaneous injection of estradiol benzoate, EB, for 11 consecutive days for a total of 315 µg, PND 0.5–10); SC30 or SC300 (single subcutaneous impant of silicone capsules containing either 30 or 300 µg EB); EBMS300 (single injection of microspheres containing 300 µg of EB. *, some animals kept until PND 335 to determine late vaginal opening (Supplementary Fig. 2). (B) Female Beagle dogs received SC9000 (silicone capsules containing 9000 µg EB) on PND 3 or 5, and their hypothalamic KISS1 expression was compared to that of control groups’ at PND 78. (C) Implants were made of silicone capsules with an inner diameter of 1.0 mm filled with EB and endcaps of silicone adhesive. (D) Biodegradable microspheres (EBMS300) consisted of matrix of poly lactic-co-glycolic acid (PLGA) forming spheres having less than 10 µm diameter and physically entrapping EB at 17.1% concentration.

Figure 2

Estradiol (E2) concentrations in serum and hypothalamus after estradiol benzoate (EB) capsule implantation. (A) E2 levels in the sera (pg/mL) from PND 0.5 to PND 30. (B) E2 levels in homogenized hypothalamic tissue (µg/mL) from PND 0.5 to PND 10. The number of animals for each date are provided in Supplementary Table 1. Error bars = SD. SC300 = 300 μg EB.

Figure 3

Neonatal estradiol benzoate (EB) inhibited reproductive organ development and ovarian function in female rats. Controls received silicone capsules with oil only. The EBx11 group received EB injections on 11 consecutive days from birth (total of 315 µg EB). The EB300 and EB30 groups received a single implant of a silicone capsule containing 300 or 30 µg EB (SC300 or SC30), respectively. (A) Estrous cyclicity in representative rats in each group from PND 62 to PND 69. E, estrus; P, proestrus; MD, met-diestrus. (B) Ovary weight/body weight ratio; uterus weight/body weight ratio by EB treatment at PND 178–211 (6–7 months), and Anovaginal distance (AVD; mm) (Control, n = 14; EBx11, n = 6; SC300, n = 10; SC30, n = 9). Error bars = SD. There was a significantly lower ovary/body weight ratio in all EB treatment groups compared to control; P-values indicate significant differences (One-way ANOVA with Tukey post hoc test). (C) Representative histology of control ovaries at PND 60–75 (2.5 months) (Control, n = 7; EBx11, n = 3; SC300, n = 4; SC30, n = 3) or PND 178–211 (6–7 months) (Control, n = 8; EBx11, n = 6; SC300, n = 5; SC30, n = 9) show numerous corpora lutea (CL). In the EB treated groups, the ovaries have no corpus luteum, but numerous anovulatory follicles (*) are present.

Figure 4

Effects of neonatal estradiol benzoate (EB) on KISS expression in the ARC region of the hypothalamus. (A) The top photo displays the AVPV and ARC regions of the hypothalamus in a sagittal section of the brain, illustrating the areas where the KISS1-immunofluorescence images were captured. In the bottom panel, serial sections from the ARC region reveal immunofluorescence for KISS1 in both control and SC300 rats on PND 29, with lower immunoreactivity in the SC300 sections. (B,C) KISS1-ir in ARC region of PND75 rats in control and SC300 (300 μg EB) groups. In B, the image of the ARC region in a rat treated with SC300 exhibited lower KISS1-ir than in the control. In C, quantification of the KISS1-ir revealed a significant decrease in the SC300 rats compared to the control group. Error bars = SD (n = 3). *P-value < 0.05 (Student’s t-test).

Figure 5

Serum testosterone and estradiol levels in adult control and SC300 (300 μg estradiol benzoate) treated rats and the effect of KP-10 injection on serum LH. Serum concentrations of (A) testosterone and (B) estradiol in PND 75 (2.5 months of age) rats (Control, n = 7; SC300, n = 4) and PND 178–211 (6–7 months of age) rats (Control, n = 6; SC300, n = 9). Significant differences between the Control and SC300 are indicated by P-values (Student’s t-test). (C). Serum LH levels in control (n = 4) and SC300 (n = 3) rats before and after the treatment of kisspeptin analog, KP-10. After intraperitoneal injection of KP-10 (50 nmole/animal) in rats at 2–2.5 months of age, serum LH levels at 30 min were significantly increased, compared to baseline before injection. Significant differences are indicated by P-values (Two-way ANOVA with Tukey post hoc test). Error bars = SD.

Figure 6

Effects of neonatal estradiol benzoate (EB) on gene expression profiles of Kiss1-clusters in Control and SC300 hypothalami. (A) The hypothalamic area used for scRNAseq is marked by dotted line. (B) Pooled hypothalamic cells of Control (4,049 total) and the SC300 group (4223 total) were UMAP clustered together. Cells were identified by marker gene expressions. Cluster 6 was used for identifying genes that were differentially expressed in SC300 and Control. (C) Down-regulated genes in the hypothalamus of SC300 compared to Control are listed with log fold-change of the average expression between the two groups (avg_logFC), percentage of cells where the feature is detected in the first group or second group (Pct. 1 or Pct. 2), P-value (p_val), and adjusted P-value (P_val_adj). (D) Down-regulated genes in SC300 are grouped according to functional categories. X-axis indicates the number of DEGs in each category. (E) The first panel shows the total number of cells in cluster 6 with Kiss1-expressing cells distinguished by orange color. The panel on the right plots the number of Kiss1-expressing cells in each group with Kiss1-expressing cells defined as Kiss1 LogExp > 0.5 (red dotted line).

Figure 7

Effects of neonatal estradiol benzoate (EB) on body and organ weights and structure of the mammary gland. Controls received silicone capsules with oil only. The EBx11 group received EB injections on 11 consecutive days from birth (total of 315 µg EB). The EB300 group received a single implant of a silicone capsule containing 300 µg EB. All organs and tissues were collected at PND 178–211 (6–7 months). (A) Body weight (BW). (B) Femur bone weight, length, and weight/length ratio. (C) The weight of essential organs is presented as the ratio of organ weight/BW. (D) Mammary gland morphology by whole mount imaging. Arrowheads indicate branching points of mammary ducts. Arrows indicate terminal end buds. Ductal end bud formation appears to be underdeveloped in the EBx11 and SC300 glands, with ductal width also being thinner than in control mammary gland tissues. Error bars = SD. Significant differences between groups are indicated by P-values (One-way ANOVA with Tukey post hoc test).

Figure 8

Neonatal injection of biodegradable estradiol benzoate-microspheres (EBMS300) inhibited vaginal opening and ovarian function. Controls were untreated females. The EBMS300 group received a single injection of 300 μg of EB in the reconstituted microspheres. (A) Vaginal opening of control and EBMS300 rats until PND 45. (B) Estrous cycle of control (n = 8) and EBMS300 (n = 5) rats from PND 44 to 50 (1.5 months). (C) Representative histology of ovaries on PND 125. In the control ovary, numerous corpora lutea (CL) are easily recognized as large homogenous structures, which resulted in a greatly enlarged organ. In the EBMS300 ovary, the CL is absent, with only anovulatory follicles (*) being present in a visibly smaller organ. (D) Volume of the ovary in control and EBMS300 rats (PND 125). Significant differences are indicated by P-values (Student’s t-test). Error bars = SD (n = 8).

Figure 9

Inhibited hypothalamic KISS1 expression and ovary development in female Beagle dogs after neonatal implant of estradiol benzoate (EB)-capsule. (A) KISS1 mRNA expression in the hypothalamus of control and SC9000 (9,000 μg EB) dogs on PND 78 (2.5 months). Error bars = SD (n = 3). P-values indicate significant differences (Student’s t-test). (B) Representative histology of control and SC9000 ovaries on PND 78 (2.5 months) (n = 3). Arrowheads and arrows indicate early primary follicles and late primary/secondary follicles, respectively.