An organism reproduces primarily to preserve and retain its species. High-quality gametes are necessary for the best possible reproduction. Infertility is a global health issue. Male factors contribute to infertility in about 50 % of couples, which makes for 8–12 % of cases globally (Vander Borght and Wyns, 2018, Bui et al., 2018, Venditti et al., 2021a, Venditti et al., 2021b). According to Slade et al. (Slade et al., 2007), infertility results in significant social and psychological trauma as well as significant financial costs for both people and healthcare systems (Wu et al., 2013). The mammalian testis creates an immunological milieu that regulates testicular function during male reproduction. According to Chen et al. (Chen et al., 2016), the testes shield immunogenic germ cells (GCs) from the negative effects of the immune system. Both systemic immune responses and local immunosuppression may be necessary to maintain testicular immunity (Gu et al., 2022). Blood-Testis-Barrier (BTB), a special barrier found inside Sertoli cells (SCs) in the testis, prevents harmful substances from passing through the seminiferous epithelium and into the adluminal compartment. Additionally, to avoid autoimmune reactions, restrict sperm antigens from entering the seminiferous epithelium (Yu et al., 2013).

The BTB is a junction between SCs; it divides seminiferous tubules into basal and apical (adluminal) compartments. BTB establishes a unique milieu favorable for germ cells (GCs) maturation and spermatogenesis (Cheng and Mruk, 2012). BTB forms in the human testis at 12–14 years of age (Cheng and Mruk, 2010) and 17–21 days postpartum in rats (Toyama et al., 2001). Millions of sperm are generated through spermatogenesis which is carried out in the testis in the seminiferous tubules (Cheng and Mruk, 2012). Spermatogenesis takes place through mitosis and meiosis in mammals, wherein the transient opening of BTB i.e., basal ectoplasmic specialization (ES) and tight junctions (TJs) leads to preleptotene spermatocytes crossing from basal to the adluminal compartment (Yan et al., 2007). The role of endogenous protein regulators in preserving the integrity of BTB has been the focus of research in recent years. Through their downstream signaling proteins at the apical and basal compartments of the testicular seminiferous epithelium, several of proteins are proteolytically cleaved by matrix metalloproteinases (MMPs) to produce peptides with biological activities that efficiently affect the BTB function (Cheng and Mruk, 2012). The laminin-γ3 derived F5-peptide via the action of MMP2 (Gao et al., 2016, Su et al., 2012, Yan et al., 2008) and collagen-α3 (IV) chain proteins derived non-collagenous 1 (NC1)-peptide via the action of MMP9 (Chen et al., 2017; Wong and Cheng, 2013) plays a significant role in disrupting the BTB. Consecutively, laminin-α2 derived LG3/4/5 peptide domain plays a crucial role in preserving the BTB integrity via proteolytic cleavage by MMP9 (Mao et al., 2020, Siu and Cheng, 2004; Wu et al., 2020). The mechanistic roles of LAMA2 in preserving BTB integrity and supporting spermatogenesis have been briefly discussed in authors previous literature (Wanjari and Gopalakrishnan, 2024).

Melatonin (MLT) is a principal indolamine neurohormone of the pineal gland. The light-dark cycles of the body and seasonal control affect its level. Because of its high lipophilicity, it can readily pass through biological membranes and reach every component of cells (Kruk et al., 2021, Tan et al., 2002). MLT has a vital role in reproductive function by regulating testicular maturation and the reproductive hormones synthesis such are GnRH, LH, and T. It prevents toxin-induced testicular damage via antioxidant activity (Li and Zhou, 2015). A study conducted on mice revealed that exposure to diquat resulted in significant testicular degeneration, sperm abnormalities, and disruption of the BTB. MLT's antioxidant and anti-apoptotic potential successfully reverse these effects (Yang et al., 2023). MLT is an established therapeutic compound that combats male infertility. The role of MLT was explored in BTB-proteins, however, its regulation is not yet explored on LAMA2, which plays a crucial role in BTB-integrity regulation.

Clomiphene (CP) citrate is a non-steroidal oral fertility drug (Patankar et al., 2007, Travison et al., 2017). It is a selective estrogen receptor modulator (SERM) that prevents the pituitary gland from receiving negative feedback from estrogen. Because of this interaction, the anterior pituitary releases more LH and FSH. Research has demonstrated that administering CP citrate has a favourable impact on serum T and sperm count in hypogonadal males with unexplained infertility (UI) (Chehab et al., 2015, Usadi and Merriam, 2015). Notably, CP citrate is widely used off-label to treat UI in males, despite the FDA's approval of the drug for the female infertility treatment (Agarwal et al., 2019, Chehab et al., 2015, Delu et al., 2020, Usadi and Merriam, 2015). According to Henriksen et al.'s recent proof-of-concept study from 2024, CP citrate application may be able to restore endogenous testosterone levels and reduce the symptoms of anabolic steroid-induced hypogonadism (ASIH) in men who have used anabolic-androgenic steroids (AAS) for an extended period. When it comes to males with ASIH, CP citrate treatment may be a safer and better option than traditional testosterone replacement therapy (TRT), particularly for young men who may have future fertility issues (Henriksen et al., 2024). Havnes et al., have conducted another proof-of-concept trial to investigate the safety of off-label CP citrate medication, whether the treatment will lessen androgen-deficient symptoms, and changes in health risks upon cessation (Havnes et al., 2024). Citrate is a salt form of drug in conjugation with CP to facilitate the pharmacokinetics and effectiveness of the CP. It does not directly contribute to the pharmacological action of CP in treating male infertility. There is no FDA-approved drug for treating male infertility, therefore, we chose CP as a conventional drug. Based on the recent literature on CP is a clinically established drug for male infertility treatment via influencing testicular function and BTB integrity through endocrine modulation. Therefore, we are trying to investigate CP’s molecular interactions in comparison with MLT on BTB-regulating protein LAMA2. Comparing both provides a pharmacological benchmark to evaluate MLT’s binding and mechanistic potential at the LAMA2 target.

Several proteins play an important role in modulating BTB structure in the testis to regulate spermatogenesis. Targeting LAMA2 protein and normalizing its level of action can help to make the BTB structure tighter. No research has attempted to investigate and identify any possible pharmacological candidate for activating LAMA2 proteins. This study explores the role of the proposed drug, MLT, compared to the conventional drug, CP, in targeting significant unexplored BTB regulatory LAMA2 protein. It uses structural biology approaches to decipher the therapeutic role of MLT in male infertility.