The usage of herbicides is constantly increasing worldwide (Benbrook, 2012; De et al., 2017; Kim et al., 2017) and their residual components and by-products may enter the soil and water have become important environmental pollutants (Choung et al., 2013; Ulber et al., 2013). Flurochloridone (FLC), (3RS, 4RS; 3RS, 4SR)-3-chloro-4-chloromethyl-1-(α, α, α-trifluoro-m-tolyl)-2-pyrrolidone, is widely used as a selective herbicide in the planting of winter wheat, sunflowers, cotton and potatoes to control a wide range of broadleaf weeds and annual grasses (Klicova et al., 2002; Theodoridis, 2006). In recent years, FLC has been increasingly used in agriculture due to its efficient removal of weeds and high safety for crops, but it also leads to the occurrence of residues in the environment (Milanova and Grigorov, 2010; EFSA, 2018). According to the report of the European Food Safety Authority (EFSA), after oral or intravenous exposure, FLC is mainly distributed in liver, kidney, testis, epididymis and blood, causing damage to male reproductive function when it acts on rats (EFSA, 2010; 2013).
Spermatogenesis is a highly coordinated complex process of proliferation and differentiation of spermatogenic cells. In mammals, spermatogenesis mainly includes spermatogonia proliferation, spermatocyte meiosis and sperm metamorphosis, which requires a specific environment and the participation of multiple factors including cells and hormones (Zhou et al., 2019; Ibtisham and Honaramooz, 2020). The proliferation of spermatogonia is the basis for the production of a large number of sperm. As a proliferation-related nuclear antigen, Ki67 is closely related to the synthesis of ribosomes during cell mitosis and can reflect cell proliferative activity, which can be used to evaluate the proliferation of spermatogonia (MacCallum and Hall, 2000; Yerushalmi et al., 2010; Sun and Kaufman, 2018). Meiosis is a strictly regulated process which is divided into two stages and essential for spermatogenesis. During the process, DNA double-strand breaks (DSBs) formation and repair are related to ensure the correct separation of homologous chromosomes (Gray and Cohen, 2016). These events are vulnerable to the interference of both internal and external factors, ensuring their correct and orderly progress is essential for spermatogenesis (Kleckner, 1996). Synaptic complex is a special structure that connects and supports homologous chromosomes during meiosis. It is composed of lateral elements and central elements, which are mainly responsible for providing a stable scaffold structure for various functional proteins in the prophase of meiosis Ⅰ. The lateral elements are composed of several lateral proteins, and its assembly is one of the markers of meiosis initiation (Petronczki et al., 2003). In mice, known components of lateral elements mainly include synaptic complex proteins SYCP3 and SYCP2 (Heyting, 1996), which can be labeled to distinguish different stages of the prophase of meiosis Ⅰ through different states of lateral elements. DSBs are the most serious form of DNA damage, and there are two main repair methods: non-homologous end joining and homologous recombination (Okayasu, 2012). Between them, homologous recombination repair, an accurate repair realized by copying sister chromatids as template (Wright et al., 2018), mainly occurs in the S/G2 phase, which is the way that carried out in programmed DSBs repair of meiosis. γH2AX protein is an important component of chromatin nucleosome histones and is crucial for the transduction of DNA damage signals, which is considered to be one of the biomarkers indicating that chromosomes are in the repair stage (Rogakou et al., 1998; Marchetti et al., 2006). Abnormal expression of γH2AX can lead to stagnation of spermatogenesis. Normally, during pachytene, autosomal repair is almost complete, while γH2AX accumulates due to the incomplete pairing of the XY chromosomes (Celeste et al., 2002; Fernandez-Capetillo et al., 2003). DMC1, first discovered in yeast, is a protein expressed during meiosis and closely related to homologous chromosome pairing and recombination (Bishop, 1994). In mice, DMC1 is expressed specifically from leptotene to zygotene, and its deletion causes the meiosis process to stop (Pittman et al., 1998; Yoshida et al., 1998). In the late stage of spermatogenesis, the morphology of round sperm cells will undergo significant changes (Yan, 2009), and abnormal sperm morphology is an important factor in the occurrence of infertility (WHO, 2010).
Testicular Sertoli cells are generally irregularly conical, extending from the base to the top of the seminiferous epithelium, which are an important part of the testicular microenvironment, providing nutrients and cytokines for germ cells. The secretion function of Sertoli cells plays an important role in the self-renewal and differentiation of spermatogonial stem cells (Meng et al., 2000; Busada et al., 2015; Griswold, 2016). At the same time, Sertoli cells have the function of phagocytosis and can phagocyte apoptotic germ cells and residual bodies, which are formed by the cytoplasm shed by differentiated sperm when they are crushed into the lumen of seminiferous tubules (Maeda et al., 2002; Nakagawa et al., 2005). When Sertoli cells are damaged, a decrease in their phagocytic capacity leads to accumulation of residual bodies in the lumen (Yefimova et al., 2008). Sertoli cells and the blood-testis barrier (BTB) formed by them are closely related to spermatogenesis and can affect meiosis (Cheng and Mruk, 2010; 2012; Chen and Liu, 2015; Gao et al., 2017). Meanwhile, they are often the targets for testicular damage caused by various environmental chemicals and result in male reproductive damage (Gao et al., 2015). GATA4, which can be used to specifically label Sertoli cells, is a key transcription factor for Sertoli cell function, regulating lactic acid metabolism and BTB function in Sertoli cells (Kyrönlahti et al., 2011; Schrade et al., 2016).
Previous research have shown that FLC has male reproductive toxicity, but the evidence is limited (Zhang et al., 2015; Liu et al., 2018). This study will be carried out from four aspects including sperm quality, spermatogenesis, toxic targets and mechanisms, aims to systematically explore the specific characteristics of FLC-induced male reproductive toxicity through a male mouse model and attends to find out the possible mechanism of its toxic effects, so as to provide more comprehensive evidence and directions of subsequent research.