Carob extract induces spermatogenesis in an infertile mouse model via upregulation of Prm1, Plzf, Bcl-6b, Dazl, Ngn3, Stra8, and Smc1b

Abstract

Ethnopharmacological relevance

Ethnopharmacological studies for drug discovery from natural compounds play an important role for developing current therapeutical platforms. Plants are a group of natural sources which have been served as the basis in the treatment of many diseases for centuries. In this regard, Ceratonia siliqua (carob) is one of the herbal medicine which is traditionally used for male infertility treatments. But so far the main mechanisms for effects of carob are unknown. Here, we intend to investigate the ability of carob extract to induce spermatogenesis in an azoospermia mouse model and determine the mechanisms that underlie its function.

Aim of the study

This is a pre-clinical animal model study to evaluate the effect of carob extract in spermatogenesis recovery.

Methods

We established an infertile mouse model with the intent to examine the ability of carob extract as a potential herbal medicine for restoration of male fertility. Sperm parameters, as well as gene expression dynamics and levels of spermatogenesis hormones, were evaluated 35 days after carob administration.

Results

Significant enhanced sperm parameters (P < 0.05) showed that the carob extract could induce spermatogenesis in the infertile mouse model. Our data suggested an anti-apototic and inducer role in the expressions of cell cycle regulating genes. Carob extract improved the spermatogenesis niche by considerable affecting Sertoli and Leydig cells (P < 0.05). The carob-treated mice were fertile and contributed to healthy offspring that matured. Our data confirmed that this extract triggered the hormonal system, the spermatogenesis-related gene expression network, and signaling pathways to induce and promote sperm production with notable level (P < 0.05). We found that the aqueous extract consisted of a polar and mainly well water-soluble substance. Carob extract might upregulate spermatogenesis hormones via its amino acid components, which were detected in the extract by liquid chromatography-mass spectrometry (LC-MS).

Conclusion

Our results strongly suggest that carob extract might be a promising future treatment option for male infertility. This finding could pave the way for clinical trials in infertile men. This is the first study that has provided reliable, strong pre-clinical evidence for carob extract as an effective candidate for fertility recovery in cancer-related azoospermia.

Introduction

Male infertility has various causes, including disruptions to the spermatogenesis process (Babakhanzadeh et al., 2020). Azoospermia is the most severe type of male infertility, and it affects 10%–20% of patients (Baker and Sabanegh Jr, 2013; Kumar, 2013). Current therapies for azoospermia include surgery techniques for sperm retrieval from the testis combined with intracytoplasmic sperm injection (ICSI) (Esteves et al., 2013). However, these techniques cannot successfully retrieve sperm in 50% of patients. Notably, the female partners of patients successfully treated by surgical means must receive hormonal therapy to become pregnant following ICSI.

Drug and hormonal therapies for azoospermia have serious adverse effects, including erectile dysfunction and mild loss of libido (Hu et al., 2018). Cardiovascular events and acceleration of prostate cancer growth (if cancer is already present) during the treatment period are adverse effects of anti-estrogen drugs such as clomifene citrate and tamoxifen, which are prescribed for azoospermic patients (Isidori et al., 2017). Therefore, it is of utmost importance to develop medications that lack adverse effects and can induce spermatogenesis in azoospermic patients and needless to use ICSI.

In this regard, Ethnopharmacology studies provides a platform for drug discovery from natural sources including plants (Sharma, 2017; Süntar, 2019). Herbal components appear to be promising alternatives to pharmaceuticals because these phytotherapeutic agents have fewer adverse effects compared to synthetic drugs. Although carob is traditionally used for male infertility (Faramarzi et al., 2019; Sharma, 2017), there is no adequate scientific basis to justify its use in the clinic. The mechanisms that underlies its function are unknown. Here, we intend to investigate the ability of carob extract to induce spermatogenesis in an azoospermic mouse model and determine the mechanisms that underlie its function. We also performed metabolite profiling of the carob extract that induced spermatogenesis in this azoospermia mouse model.