The Sry gene on the Y chromosome is essential for differentiating mammalian testes from bipotential genital ridges, as demonstrated by Koopman et al. [1]. Transient expression of Sry in a small subset of genital crest cells is sufficient to initiate male gonad development [[2], [3], [4], [5]]. Sry activates specific genes, including Sox9, which are crucial in testicular development [6]. Inadequate Sry expression can lead to abnormal testis development in XY embryos [7], highlighting its importance in disorders related to sexual development and infertility [8]. Beyond embryonic development, Sry is also expressed in the testes of adult mammals, including mice, humans, and marsupials [[9], [10], [11]]. This adult expression in germ cells suggests a role in testicular physiology [12]. Research is ongoing to determine how much Sry is expressed during spermatogenesis in adult animals [11,13,14].

The Sry gene produces two forms of transcripts: linear mRNA and circular mRNA, both present in the testes of adult mice [[11], [12], [13],15]. Adult mice present a relative higher expression of circular mRNA than the linear one [11]. The functional significance of these transcripts in adult testes, particularly in germ cell survival and fertility, is a subject of current investigation [11]. Meiotic and post-meiotic germ cells were shown to contain circular Sry RNA, which is most highly expressed in round spermatids [11]. In spermatogonia, mature sperm, or Sertoli cells, circular Sry RNA was not found [11]. The circular RNA is believed to participate in the intricate regulatory network by competing for interactions with microRNAs and RNA-binding proteins [11].

Gene silencing approaches, such as small interfering RNAs (siRNAs), are frequently utilized to verify the function of particular RNAs [16]. Transfecting siRNA into cells can be done in different ways, such as using chemical transfection (Lipofectamine), viral-mediated delivery (expression vectors), mechanical transfection (electroporation), and nanoparticle delivery [16,17]. In this study, carbon nanotubes combined with siRNA targeting the Sry RNA are used to investigate the effects of post-transcriptional inhibition [18]. Carbon nanotubes are selected for their needle-like shape and ability to cross cellular membranes, efficiently delivering siRNA into cells. This method offers an alternative to conditional transgenic mouse models [11]. This approach enables a detailed investigation of Sry mRNA and its impact on the cellular processes of adult testicular cells [[19], [20], [21]].

In this context, our objective was to evaluate the impact of systemically administered siRNA targeting the circular form of the Sry RNA on the structure and function of the testicles. We also looked into the presence and potential outcomes of carbon nanotubes, used to carry siRNA, inside the testes. We measured variations in testicular blood flow, examined changes in the tubular and intertubular compartments, and evaluated the histology of the epididymal duct. The research aimed to shed more light on how the Sry RNA silencing affected the health of male reproduction.