Carob extract induces spermatogenesis in an infertile mouse model via upregulation of Prm1, Plzf, Bcl-6b, Dazl, Ngn3, Stra8, and Smc1b
Graphical abstract
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.
Section snippets
Plant material
Carob has distinguishing characteristics that are not confused with other species. Carob pods are quite special from the genus Ceratonia. Even other species of this genus do not have carob-like pods (Ceratonia siliqua). We bought these pods from a reputable herbal store. The pods of dried carob (Ceratonia siliqua), which is a well-known medicinal plant (GRUNER, 1930; MH, 1844; Stansbury, 2019), were purchased from a reputable herbal store and identified by Dr. Ayyari, Department of
Carob extract profile
A final brownish powder from the carob pod aqueous extract was obtained after freeze drying, and the yield of the aqueous extract was 9.37%. As expected, the aqueous extract consisted of a polar and mainly well water-soluble substance. The peaks were mostly composed of carbohydrates and some organic acids. These compounds, as well as diverse ester derivatives of disaccharides, were present in the extract. The first three chromatographic peaks consisted of a dihexose(s), a hexose(s),
Discussion
The medicinal use of herbs has captivated the attention of experts for the treatment of reproductive disorder (Sharma et al., 2016) because of their decreased adverse effects compared to chemical drugs and their compatibility with human physiology (Hossen et al., 2016; Sharma et al., 2016).
Our findings of the carob ingredients including phenolics were consistent with the literature (Farag et al., 2019; Owis and El-Naggar, 2016; Papagiannopoulos et al., 2004). Additional phenolic compounds
Conclusion
Based on the findings of this study, carob extract induces spermatogenesis in an infertile mouse model by affecting genes involved in spermatogenesis, activating the GDNF, TGF-β, and BMP4 signaling pathways and targeting the hormonal system. Our study revealed an anti-apoptotic role for carob extract in testes and activation of cell cycle regulating gene expressions. The efficacy of this herb in the infertile human testis has yet to be determined; thus, clinical trials should be conducted to
Authors' contributions
Z.G.: Collection and data assembly, data analysis and interpretation, manuscript writing. A.E.: Contributed to data collection. M.G.: Performed gene expression experiments for mice that received the carob extract. M.A.: Prepared the carob extract. M.A., J.Z., and M.K.: Performed the experiments and data analysis to prepare a metabolic profile of the extract. H.B.: Data analysis and interpretation, and manuscript writing. P.E. and F.E.: Study conception and design, data analysis and
Funding
This study was supported by a grant from Royan Institute (grant no. 96000255) to F.E. and P.E.
CRediT authorship contribution statement
Zeynab Ghorbaninejad: Data Collection and assembly, data analysis and interpretation, manuscript writing. Atiyeh Eghbali: Contributed to data collection, , administrative and financial support. Mahsa Ghorbaninejad: Performed gene expression experiments. Mahdi Ayyari: Prepared the carob extract, Performed the experiments and data analysis to prepare a metabolic profile of the extract. Jerzy Zuchowski: Performed the experiments and data analysis to prepare a metabolic profile of the extract.
Declaration of competing interest
The authors declare that they do not have any conflicts of interest.
Acknowledgements
We would like to express our appreciation to Dr. Rabientaj for her advice on determining the concentrations of the extract and Miss Forough Azam Sayahpour for her kind support during the gene expression experiments.
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