Infertility has become one of the most serious diseases worldwide, and 50% of this disease can be attributed to male-related factors. Spermatogenesis, by definition, is a complex process by which spermatogonial stem cells (SSCs) self-renew to maintain stem cell population within the testes and differentiate into mature spermatids. It is of great significance to uncover gene regulation and signaling pathways that are involved in the fate determinations of SSCs with aims to better understand molecular mechanisms underlying human spermatogenesis and identify novel targets for gene therapy of male infertility. Significant achievement has recently been made in demonstrating the signaling molecules and pathways mediating the fate decisions of mammalian SSCs. In this review, we address key gene regulation and crucial signaling transduction pathways in controlling the self-renewal, differentiation, and apoptosis of SSCs, and we illustrate the networks of genes and signaling pathways in SSC fate determinations. We also highlight perspectives and future directions in SSC regulation by genes and their signaling pathways. This review could provide novel insights into the genetic regulation of normal and abnormal spermatogenesis and offer molecular targets to develop new approaches for gene therapy of male infertility.
Humans ; Male ; Signal Transduction/physiology* ; Apoptosis/physiology* ; Spermatogenesis/physiology* ; Cell Differentiation ; Adult Germline Stem Cells/physiology* ; Spermatogonia/cytology* ; Gene Expression Regulation ; Animals ; Infertility, Male/genetics* ; Cell Self Renewal/genetics*

Icariin is a pure compound derived from Epimedium brevicornu Maxim, and it helps the regulation of male reproduction. Nevertheless, the role and underlying mechanisms of Icariin in mediating male germ cell development remain to be clarified. Here, we have demonstrated that Icariin promoted proliferation and DNA synthesis of mouse spermatogonial stem cells (SSCs). Furthermore, surface plasmon resonance iron (SPRi) and molecular docking (MOE) assays revealed that phosphodiesterase 5A (PDE5A) was an important target of Icariin in mouse SSCs. Mechanically, Icariin decreased the expression level of PDE5A. Interestingly, hydrogen peroxides (H 2 O 2 ) enhanced the expression level of phosphorylation H2A.X (p-H2A.X), whereas Icariin diminished the expression level of p-H2A.X and DNA damage caused by H 2 O 2 in mouse SSCs. Finally, our in vivo animal study indicated that Icariin protected male reproduction. Collectively, these results implicate that Icariin targets PDE5A to regulate mouse SSC viability and DNA damage and improves male reproductive capacity. This study thus sheds new insights into molecular mechanisms underlying the fate decisions of mammalian SSCs and offers a scientific basis for the clinical application of Icariin in male reproduction.
Male ; Animals ; Flavonoids/pharmacology* ; Mice ; Cyclic Nucleotide Phosphodiesterases, Type 5/drug effects* ; DNA Damage/drug effects* ; Cell Survival/drug effects* ; Cell Proliferation/drug effects* ; Spermatogonia/drug effects* ; Reproduction/drug effects* ; Adult Germline Stem Cells/metabolism* ; DNA Replication/drug effects*

Infertility has become a serious disease since it affects 10%-15% of couples worldwide, and male infertility contributes to about 50% of the cases. Notably, a significant decrease occurs in the newborn population by 7.82 million in 2020 compared to 2016 in China. As such, it is essential to explore the effective methods of obtaining functional male gametes for restoring male fertility. Stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), spermatogonial stem cells (SSCs), and mesenchymal stem cells (MSCs), possess the abilities of both self-renewal and differentiation into germ cells. Significantly, much progress has recently been achieved in the generation of male germ cells in vitro from various kinds of stem cells under the specified conditions, e.g., the coculturing with Sertoli cells, three-dimensional culture system, the addition of growth factors and cytokines, and/or the overexpression of germ cell-related genes. In this review, we address the current advance in the derivation of male germ cells in vitro from stem cells based on the studies of the peers and us, and we highlight the perspectives and potential application of stem cell-derived male gametes in reproductive medicine.
Humans ; Infant, Newborn ; Male ; Germ Cells ; Embryonic Stem Cells ; Cell Differentiation ; Infertility, Male ; Induced Pluripotent Stem Cells

Male ; Humans ; Avena ; Infertility, Male/genetics* ; Oligospermia/genetics* ; Asthenozoospermia ; Spermatozoa

Spermatogonial stem cells (SSCs) have great applications in both reproductive and regenerative medicine. Primates including monkeys are very similar to humans with regard to physiology and pathology. Nevertheless, little is known about the isolation, the characteristics, and the culture of primate SSCs. This study was designed to identify, isolate, and culture monkey SSCs. Immunocytochemistry was used to identify markers for monkey SSCs. Glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRA1)-enriched spermatogonia were isolated from monkeys, namely Macaca fascicularis (M. fascicularis), by two-step enzymatic digestion and magnetic-activated cell sorting, and they were cultured on precoated plates in the conditioned medium. Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and RNA sequencing were used to compare phenotype and transcriptomes in GFRA1-enriched spermatogonia between 0 day and 14 days of culture, and xenotransplantation was performed to evaluate the function of GFRA1-enriched spermatogonia. SSCs shared some phenotypes with rodent and human SSCs. GFRA1-enriched spermatogonia with high purity and viability were isolated from M. fascicularis testes. The freshly isolated cells expressed numerous markers for rodent SSCs, and they were cultured for 14 days. The expression of numerous SSC markers was maintained during the cultivation of GFRA1-enriched spermatogonia. RNA sequencing reflected a 97.3% similarity in global gene profiles between 0 day and 14 days of culture. The xenotransplantation assay indicated that the GFRA1-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-Kit

OBJECTIVE:To establish the quality standard of Chushi pill. METHODS:Microscopic identification and TLC were adopted for the qualitative identification of Cortex moutan,C. dictamni,Angelica sinensis,Rubia cordifolia and Gardeniae fructus in Chushi pill;HPLC was performed to determine the contents of paeonol and baicalin. It was performed on column of Kro-masil 100-5 C18 with mobile phase of methanol-water-phosphoric acid(47∶53∶0.2,V/V/V)at the flow rate of 1.0 ml/min,the detec-tion wavelength was 280 nm,the temperature was 25 ℃ and the volume was 10 μl. RESULTS:The microscopic identification showed microscopic characteristics of C. moutan and C. dictamni,and characteristics of A. sinensis,R. cordifolia and G. fructus were identified by TLC;the linear range of paeonol was 0.106 24-2.124 8 μg(r=0.999 9)and baicalin was 0.059 04-1.180 8 μg (r=0.999 9);RSDs of precision,stability and reproducibility tests were no more than 2.06%;average recoveries were respective-ly 101.56%(RSD=1.68%,n=9)and 100.16%(RSD=1.13%,n=9). CONCLUSIONS:The method is simple,accurate and re-producible,and can be used for the quantity control of Chushi pill.

To investigate the effects of gossypol acetic acid (GA) on proliferation and apoptosis of the macrophage cell line RAW264.7 and further understand the possible underlying mechanism responsible for GA-induced cell apoptosis, RAW264.7 cells were treated with GA (25~35 micromol/L) for 24 h and the cytotoxicity was determined by MTT assay, while apoptotic cells were identified by TUNEL assay, acridine orange/ethidium bromide staining and flow cytometry. Moreover, mitochondrial membrane potential (DeltaPsi(m)) with Rhodamine 123 and reactive oxygen species (ROS) with DCFH-DA were analyzed by fluorescence spectrofluorometry. In addition, the expression of caspase-3 and caspase-9 was assessed by Western Blot assay. Finally, the GA-induced cell apoptosis was evaluated by flow cytometry in the present of caspase inhibitors Z-VAD-FMK and Ac-LEHD-FMK, respectively. GA significantly inhibited the proliferation of RAW264.7 cells in a dose-dependent manner, and caused obvious cell apoptosis and a loss of DeltaPsi(m) in RAW264.7 cells. Moreover, the ROS production in cells was elevated, and the levels of activated caspase-3 and caspase-9 were up-regulated in a dose-dependent manner. Notably, GA-induced cell apoptosis was markedly inhibited by caspase inhibitors. These results suggest that GA-induced RAW264.7 cell apoptosis may be mediated via a caspase-dependent mitochondrial signaling pathway.
Animals ; Antineoplastic Agents, Phytogenic/*pharmacology ; Apoptosis/*drug effects ; Cell Line ; Cell Proliferation/*drug effects ; Dose-Response Relationship, Drug ; Gossypol/*analogs & derivatives/pharmacology ; Membrane Potential, Mitochondrial/*drug effects ; Mice ; Mice, Inbred BALB C ; Reactive Oxygen Species/*metabolism ; Signal Transduction/*drug effects

OBJECTIVETo establish the chromatographic fingerprints for the anti-tumor flavonoids of Caulis spatholobi (SSCE). It could used to reflect the chemical information in this part comprehensively, and identify the chemical consitituents preliminarily.

METHODThe HPLC-DAD analysis method was performed on the column Kromasil 100-5PHENYL (4.6 mm x 250 mm, 5 microm). The mobile phase was water (0.5% acetic acid)- methanol in gradient elution and the detection wavelength was 254 nm.

RESULTThe chromatographic fingerprint of SSCE was established, which showed 16 characteristic peaks from 10 batches of medicinal materials. Among them, the peaks 1, 3, 4, 5, 8, 9, 10, 12, 13, and 16 were identified 3,4-dihodroxybenzoic acid, 4-Hydroxybenzoic Acid, epicatechin, puerarin, daidzein, liquiritigenin, calycosin, genistein, formononetin, and prunetin, respectively.

CONCLUSIONThe method is convenient, reproducibility and stability. It can used for quality control of the anti-tumor flavonoids of C. spatholobi (SSCE).

Antineoplastic Agents ; analysis ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; analysis ; chemistry ; Fabaceae ; chemistry ; metabolism ; Flavonoids ; analysis ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Reproducibility of Results