Yi-Xun Liu

Scrotal heat stress causes a transient alteration in tight junctions and induction of TGF-β expression

Specialized junctions, which occur at sites of Sertoli-Sertoli and Sertoli-germ cell contact of seminiferous epithelium, play pivotal roles in spermatogenesis. Slight increase in scrotal temperature can induce oligospermia or azoospermia via increasing germ cell apoptosis. In this study, we demonstrated that the expression of tight junction (TJ) components, such as occludin, claudin-3 and zonula occludens-1 (ZO-1), was reduced 24-48h after a single mild scrotal heat exposure (43°C for 30min), whereas mRNA levels of claudin-11 were increased. Moreover, the protein localization of occludin and ZO-1 was lost from the blood-testis barrier (BTB) site, whereas claudin-11 immunostaining became diffuse and cytoplasmic 2days following heat exposure. Electron microscopic analysis showed that 2days after the heat treatment, the intercellular space between the two adjacent Sertoli cells was expanded, coupled with defragmentation of actin bundles and the endoplasmic reticulum. In addition, the TJ permeability increased significantly 2days after the heat exposure and recovered approximately 10days later. Heat-induced reversible BTB disruption was associated with a transient induction of transforming growth factor (TGF)-β2, -3 and p38 mitogen-activated protein kinase activation. However, the TGF-β antagonist only partially prevented the heat-induced BTB disruption. In conclusion, the expression of TJ-associated molecules and BTB were reversibly perturbed after mild testicular hyperthermia, and the induction of TGF-β expression may be partially involved in heat-induced BTB damage.

Loss of Gata4 in Sertoli cells impairs the spermatogonial stem cell niche and causes germ cell exhaustion by attenuating chemokine signaling

Sertoli cells, the primary somatic cell in the seminiferous epithelium, provide the spermatogonial stem cell (SSC) microenvironment (niche) through physical support and the expression of paracrine factors. However, the regulatory mechanisms within the SSC niche, which is primarily controlled by Sertoli cells, remain largely unknown. GATA4 is a Sertoli cell marker, involved in genital ridge initiation, sex determination and differentiation during the embryonic stage. Here, we showed that neonatal mice with a targeted disruption of Gata4 in Sertoli cells (Gata4flox/flox; Amh-Cre; hereafter termed Gata4 cKO) displayed a loss of the establishment and maintenance of the SSC pool and apoptosis of both gonocyte-derived differentiating spermatogonia and meiotic spermatocytes. Thus, progressive germ cell depletion and a Sertoli-cell-only syndrome were observed as early as the first wave of murine spermatogenesis. Transplantation of germ cells from postnatal day 5 (P5) Gata4 cKO mice into KitW/W-v recipient seminiferous tubules restored spermatogenesis. In addition, microarray analyses of P5 Gata4 cKO mouse testes showed alterations in chemokine signaling factors, including Cxcl12, Ccl3, Cxcr4 (CXCL12 receptor), Ccr1 (CCL3 receptor), Ccl9, Xcl1 and Ccrl2. Deletion of Gata4 in Sertoli cells markedly attenuated Sertoli cell chemotaxis, which guides SSCs or prospermatogonia to the stem cell niche. Finally, we showed that GATA4 transcriptionally regulated Cxcl12 and Ccl9, and the addition of CXCL12 and CCL9 to an in vitro testis tissue culture system increased the number of PLZF+ undifferentiated spermatogonia within Gata4 cKO testes. Together, these results reveal a novel role for GATA4 in controlling the SSC niche via the transcriptional regulation of chemokine signaling shortly after birth.

Development, function and fate of fetal Leydig cells.

During fetal testis development, fetal Leydig cells (FLCs) are found to be originated from multiple progenitor cells. FLC specification and function are under tight regulation of specific genes and signaling proteins. Furthermore, Sertoli cells play a crucial role to regulate FLC differentiation during fetal testis development. FLC progenitor- and FLC-produced biomolecules are also involved in the differentiation and activity of rodent FLCs. The main function of FLCs is to produce androgens to masculinize XY embryos. However, FLCs are capable of producing androstenedione but not testosterone due to the lack of 17β-HSD (17β-hydroxysteroid dehydrogenase), but fetal Sertoli cells express 17β-HSD which thus transforms androstenedione to testosterone in the fetal testis. On the other hand, FLCs produce activin A to regulate Sertoli cell proliferation, and Sertoli cells in turn modulate testis cord expansion. It is now generally accepted that adult Leydig cells (ALCs) gradually replace FLCs during postnatal development to produce testosterone to support spermatogenesis as FLCs undergo degeneration in neonatal and pre-pubertal testes. However, based on studies using genetic tracing mouse models, FLCs are found to persist in adult testes, making up ∼20% of total Leydig cells. In this review, we evaluate the latest findings regarding the development, function and fate of FLCs during fetal and adult testis development.

Wnt/β-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components.

Wnt signaling is an evolutionarily conserved pathway that regulates cell proliferation, differentiation and apoptosis. To investigate the possible role of Wnt signaling in the regulation of ovarian follicular development, secondary follicles were isolated and cultured in vitro in the presence or absence of its activator (LiCl or Wnt3a) or inhibitor (IWR-1). We have demonstrated that activation of β-catenin signals by activators dramatically suppressed follicular development by increasing granulosa cell apoptosis and inhibiting follicle steroidogenesis. In contrast, inhibition of Wnt signaling by IWR-1 was observed with better developed follicles and increased steroidogenesis. Further studies have shown that the transcription factor Forkhead box O3a (Foxo3a) and its downstream target molecules were modulated by the activators or the inhibitor. These findings provide evidence that Wnt signaling might negatively regulate follicular development potentially through Foxo3a signaling components.

Wt1 deficiency causes undifferentiated spermatogonia accumulation and meiotic progression disruption in neonatal mice

Spermatogenesis is a complex process involving the regulation of multiple cell types. As the only somatic cell type in the seminiferous tubules, Sertoli cells are essential for spermatogenesis throughout the spermatogenic cycle. The Wilms tumor gene, Wt1, is specifically expressed in the Sertoli cells of the mouse testes. In this study, we demonstrated that Wt1 is required for germ cell differentiation in the developing mouse testes. At 10 days post partum, Wt1-deficient testes exhibited clear meiotic arrest and undifferentiated spermatogonia accumulation in the seminiferous tubules. In addition, the expression of claudin11, a marker and indispensable component of Sertoli cell integrity, was impaired in Wt1(-/flox); Cre-ER(TM) testes. This observation was confirmed in in vitro testis cultures. However, the basal membrane of the seminiferous tubules in Wt1-deficient testes was not affected. Based on these findings, we propose that Sertoli cells status is affected in Wt1-deficient mice, resulting in spermatogenesis failure.

Androgen receptor in Sertoli cells regulates DNA double-strand break repair and chromosomal synapsis of spermatocytes partially through intercellular EGF-EGFR signaling

Spermatogenesis does not progress beyond the pachytene stages of meiosis in Sertoli cell-specific AR knockout (SCARKO) mice. However, further evidence of meiotic arrest and underlying paracrine signals in SCARKO testes is still lacking. We utilized co-immunostaining of meiotic surface spreads to examine the key events during meiotic prophase I. SCARKO spermatocytes exhibited a failure in chromosomal synapsis observed by SCP1/SCP3 double-staining and CREST foci quantification. In addition, DNA double-strand breaks (DSBs) were formed but were not repaired in the mutant spermatocytes, as revealed by γ-H2AX staining and DNA-dependent protein kinase (DNA-PK) activity examination. The later stages of DSB repair, such as the accumulation of the RAD51 strand exchange protein and the localization of mismatch repair protein MLH1, were correspondingly altered in SCARKO spermatocytes. Notably, the expression of factors that guide RAD51 loading onto sites of DSBs, including TEX15, BRCA1/2 and PALB2, was severely impaired when either AR was down-regulated or EGF was up-regulated. We observed that some ligands in the epidermal growth factor (EGF) family were over-expressed in SCARKO Sertoli cells and that some receptors in the EGF receptor (EGFR) family were ectopically activated in the mutant spermatocytes. When EGF-EGFR signaling was repressed to approximately normal by the specific inhibitor AG1478 in the cultured SCARKO testis tissues, the arrested meiosis was partially rescued, and functional haploid cells were generated. Based on these data, we propose that AR in Sertoli cells regulates DSB repair and chromosomal synapsis of spermatocytes partially through proper intercellular EGF-EGFR signaling.

Melatonin reduces oxidative damage and upregulates heat shock protein 90 expression in cryopreserved human semen

Abstract Sperm cells can be damaged during the semen cryopreservation process, decreasing their fertilizing ability. Physical damage and oxidative stress may occur during the freeze–thawing process. Antioxidants such as the native antioxidant melatonin can potentially improve cryopreservation outcomes. In this study, we added melatonin to cryoprotectant to examine its effect on frozen–thawed human sperm. We found that adding 0.1 mM melatonin to cryoprotectant significantly increased sperm viability (24.80 ± 0.46% vs. 20.97 ± 1.27%, P < 0.05) and membrane integrity (P < 0.05), and decreased intracellular reactive oxygen species and lipid peroxidation damage. Furthermore, mRNA levels of the transcription factor NF‐E2‐related factor‐2 and its downstream genes were significantly increased. Resistance to oxidative stress was enhanced and expression of the antiapoptotic gene Bcl‐2 was increased by inclusion of 0.1 mM melatonin in the cryoprotectant. Moreover, 0.1 mM melatonin upregulated the expression of heat shock protein 90 (HSP90), which confers resistance to stressors in frozen–thawed sperm. Results obtained upon addition of inhibitors of melatonin receptors (luzindole and 4‐P‐PDOT) and an HSP90 inhibitor (geldanamycin) in the cryoprotectant demonstrated that melatonin promoted HSP90 translation via the melatonin receptor MT1 and increased adenosine triphosphate levels, thus increasing the viability of thawed sperm. HighlightsMelatonin increases motility and membrane integrity of frozen–thawed sperm.Melatonin decreases the ROS content of frozen–thawed human sperm.Melatonin promotes HSP90 expression in frozen–thawed human sperm via MT1.

Melatonin promotes development of haploid germ cells from early developing spermatogenic cells of Suffolk sheep under in vitro condition

Promotion of spermatogonial stem cell (SSC) differentiation into functional sperms under in vitro conditions is a great challenge for reproductive physiologists. In this study, we observed that melatonin (10−7 m) supplementation significantly enhanced the cultured SSCs differentiation into haploid germ cells. This was confirmed by the expression of sperm special protein, acrosin. The rate of SSCs differentiation into sperm with melatonin supplementation was 11.85 ± 0.93% which was twofold higher than that in the control. The level of testosterone, the transcriptions of luteinizing hormone receptor (LHR), and the steroidogenic acute regulatory protein (StAR) were upregulated with melatonin treatment. At the early stage of SSCs culture, melatonin suppressed the level of cAMP, while at the later stage, it promoted cAMP production. The similar pattern was observed in testosterone content. Expressions for marker genes of meiosis anaphase, Dnmt3a, and Bcl‐2 were upregulated by melatonin. In contrast, Bax expression was downregulated. Importantly, the in vitro‐generated sperms were functional and they were capable to fertilize oocytes. These fertilized oocytes have successfully developed to the blastula stage.

Toll-Like Receptor 4 Promotes NO Synthesis by Upregulating GCHI Expression under Oxidative Stress Conditions in Sheep Monocytes/Macrophages.

Many groups of Gram-negative bacteria cause diseases that are harmful to sheep. Toll-like receptor 4 (TLR4), which is critical for detecting Gram-negative bacteria by the innate immune system, is activated by lipopolysaccharide (LPS) to initiate inflammatory responses and oxidative stress. Oxidation intermediates are essential activators of oxidative stress, as low levels of free radicals form a stressful oxidative environment that can clear invading pathogens. NO is an oxidation intermediate and its generation is regulated by nitric oxide synthase (iNOS). Guanosine triphosphate cyclohydrolase (GCHI) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, which is essential for the production of inducible iNOS. Previously, we made vectors to overexpress the sheep TLR4 gene. Herein, first generation (G1) of transgenic sheep was stimulated with LPS in vivo and in vitro, and oxidative stress and GCHI expression were investigated. Oxidative injury caused by TLR4 overexpression was tightly regulated in tissues. However, the transgenic (Tg) group still secreted nitric oxide (NO) when an iNOS inhibitor was added. Furthermore, GCHI expression remained upregulated in both serum and monocytes/macrophages. Thus, overexpression of TLR4 in transgenic sheep might accelerate the clearance of invading microbes through NO generation following LPS stimulation. Additionally, TLR4 overexpression also enhances GCHI activation.

Erratum to: Over-expression of Toll-like receptor 2 up-regulates heme oxygenase-1 expression and decreases oxidative injury in dairy goats

[This corrects the article DOI: 10.1186/s40104-016-0136-2.].