Retinoic acid: A potential therapeutic agent for cryptorchidism infertility based on investigation of flutamide-induced cryptorchid rats in vivo and in vitro
Introduction
Cryptorchidism is defined as one or both testicles absent from the scrotum. This often causes non-obstructive infertility in the male, even after orchiopexy [1]. The rate of infertility in cryptorchidism is ˜89% [2], which has been increasing due to rising numbers of premature neonates and low-birth-weight infants, and environmental pollution [3].
The etiology of cryptorchidism remains unclear, but it is well known that the integrity of the blood-testis-barrier (BTB, also known as the Sertoli cell barrier) is compromised in these patients [4]. Methods to investigate cryptorchidism-induced infertility are a major concern of research in andrology.
Flutamide is an anti-androgen drug that metabolizes into 2-hydroxyflutamide in the liver, and then blocks the androgen receptor. Flutamide has also been used to inhibit spermatogenesis in animal experiments, and to establish a cryptorchid animal model in offspring during latter pregnancy [5]. Because flutamide intervenes during the fetal stage, compared with other modeling methods such as surgery the etiology of cryptorchidism in offspring is more similar to the pathological mechanism in humans. In our previous study, we successfully established a cryptorchid model in rats using flutamide [6].
Methods used to study BTB function and normal spermatogenesis in vitro have included Sertoli and Leydig cell cultures, and culture of some spermatogonial stem cells [7,8]. To improve replication of the complicated process of BTB maturation, we have utilized an organotypic culture system. Many studies have shown that this culture method is reliable and practicable; the formation of the BTB in this in vitro model matches the pace of BTB formation in both animals and humans in vivo [9,10].
Retinoic acid (RA) is a metabolite of vitamin A, and required for normal meiosis. RA signaling has been reported to mediate precocious spermatogonial differentiation and meiotic initiation, and contributes to particular seminiferous stages [11]. Rats with vitamin A deficiency show deformity of the genitourinary tract, leading to arrested spermatogenesis. Several studies have indicated that vitamin A or RA is crucial for normal spermatogenesis [12,13]. A recent study indicated that RA could induce mouse spermatogonial stem cells to develop into zygotene spermatocytes [14]. In addition, RA contributes to periodic organization of the BTB by altering the expressions of stage-dependent genes in Sertoli cells [15]. However, few studies have examined an association between RA and formation of the BTB in cryptorchidism.
The BTB comprises three types of junctions: tight, gap, and anchoring. The tight junction is the major component. By forming a selective barrier between the Sertoli cells of the seminiferous tubule, the testes are isolated from the circulation system, and the proper microenvironment for continuous generation of spermatozoa is maintained [16]. Dysfunction of the spermatogenic microenvironment severely compromises spermatogenesis [17].
Proteins that are essential for maintaining the function of the tight junction include occludin, zonula occludens (ZO-1), N-cadherin, and claudin-11 (Cld11). Connexin 43 (Cx43) is a gap junction marker that is necessary for normal testis development and spermatogenesis, and regulates the formation of the BTB [18]. In the present study, we tested the hypothesis that RA may help ameliorate dysfunction of the BTB via regulation of these important proteins. To test this hypothesis, the effect of RA for improving spermatozoon quality was investigated in vivo in a cryptorchid rat model induced with flutamide. Furthermore, an association between RA and levels of these proteins was investigated in vitro and in vivo.
Section snippets
Animals and treatments
Female Sprague-Dawley rats (250–300 g) from the Experimental Animal Center of Chongqing Medical University (SCXK 2018-0003) were housed in the Animal Center of Chongqing Children’s Hospital (SYXK 2017-0012). All animals had free access to food and tap water, and housed in a pathogen-free environment (12-h light/12-h dark; 25 ± 2 ℃; humidity 55 ± 5%). The females were paired with males overnight and checked for a vaginal sperm plug in the morning, which was designated as embryonic day (ED)0.5.
Verification of the flutamide-induced cryptorchid model in male pups
Flutamide, delivered via subcutaneous injections to pregnant rats in groups Flu-only and Flu + RA, was used to induce a cryptorchid model in male offspring. The rates cryptorchidism rates were significantly higher in the Flu-only and Flu + RA groups compared with the control, while the Flu-only and Flu + RA groups were similar. The number of pups with unilateral cryptorchidism and bilateral cryptorchidism is shown in Table 1. In PND30 cryptorchid rats, affected side testicle did not fully
Discussion
In the present study, we investigated if RA alone could improve spermatozoon quality by restoring BTB formation in flutamide-induced cryptorchidism and dyszoospermia in both in vivo and in vitro models.
Vitamin A deficiency can lead to many subsequent health problems, even death. Worldwide, it is still a major public health problem troubling about 190 million children under the age of five [28]. In recent years, the necessity of supplementing vitamin A for children has been questioned, because
Author contributions
Wei Guanghui, Zhang Deying, and Lin Tao conceptualized the experiments. Zhang Deying, Shen Lianju, Long Chunlan, and Wei Guanghui are responsible for the experimental design. Hu Dong, Liu Bo, Peng Jinpu, Yu Yihang, and Zhou Yu conducted the data analysis. Zhou Yu and Zhang Deying wrote the manuscript. Wei Guanghui, Tao Xu, Yuanyuan Zhang, Peter Timashev, and He Dawei revised the manuscript. All authors read, commented on, and approved the final manuscript.
Funding
The present study was financially supported by the National Natural Science Foundation of China (Grant No. 81771566), the Entrepreneurship and Innovation Support Program for Returned Overseas Chinese Scholars, Chongqing (cx2017015), and the Research and Innovation Project of Chongqing Graduate Students (Grant No. CYS17165).
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
We thank the participants in this study. Thanks to Yuanyuan Zhang of Wake Forest University who edited an initial version of this manuscript.
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