# Testicular Gap (CX43) and Tight Junction (OCLN, CLDN3, 5 and 11) Components in the Dog Are Affected by GnRH-Mediated Downregulation

## TL;DR

GnRH implants in male dogs temporarily disrupt testicular junctions needed for sperm production, but these structures recover after implant removal.

## Contribution

This study shows that GnRH implants reversibly affect testicular junction proteins and mRNA in dogs, impacting the blood-testis barrier.

## Key findings

- GnRH implants disrupt the blood-testis barrier by altering CX43 and CLDN11 distribution in Sertoli cells.
- mRNA expressions of OCLN, CLDN5, and CX43 differ significantly during recovery compared to untreated adult dogs.
- CLDN11 and CX43 protein staining indicates barrier disruption during treatment and recovery by week 9.

## Abstract

Slow-release gonadotropin-releasing hormone (GnRH)-agonist implants are used as a medical alternative to surgical castration, reversibly inducing basal testosterone and infertility in male dogs, with full recovery of testicular functions subsequent to implant removal. We hypothesized that the blood–testis barrier, essential for normal spermatogenesis, is reversibly affected by treatment. Gap and tight junction component expressions were studied at mRNA and protein level during efficient treatment and different weeks of recovery following implant removal and compared to untreated adult dogs. In relation to treatment, the blood–testis barrier was disrupted but recovered following recovery of spermatogenesis.

Following the downregulation of testicular endocrine and germinative function by slow-release gonadotropin-releasing hormone (GnRH)-agonist implants, testicular functions are quickly restored after implant removal. As an intact blood–testis barrier (BTB) is crucial for normal spermatogenesis and its integrity is FSH- and androgen-dependent, alterations in the BTB gene and protein expressions during downregulation and subsequent restart seem inevitable. We investigated occludin (OCLN), claudin (CLDN) 3, 5, 11, and connexin (CX) 43 mRNA-, and CLDN11 and CX43 protein expressions during GnRH implant-induced downregulation (W0) and restart of spermatogenesis after implant removal (week, W, 3–12). Untreated juvenile (JG) and adult dogs (CG) served as controls. Sertoli cells were significantly affected by treatment (reduced nuclear area, OCLN, and CLDN5 expressions). All investigated genes (except CLDN3) differed significantly during restart (W0–12) compared with CG (p < 0.05). CLDN11 and CX43 immunopositive staining was absent or diffuse cytoplasmic at downregulation and relocated at W9, indicating disruption and subsequent restorage of BTB. As W0 and JG differed considerably, our results suggest that the model cannot mimic puberty. In conclusion, GnRH implant-induced long-term gonadotropin suppression disrupts testicular CX43 and CLDN11 distribution and changes gap and tight junction mRNA expression. Treatment effects are reversible suggesting re-establishment of the BTB.