[Expression of the Ces5a gene in the rat testis].

OBJECTIVE: To study the expression of the Ces5a gene in the development of the rat testis. METHODS: Using RT-PCR, Western blot, immunohistochemistry and HE staining, we determined the mRNA transcription level, protein expression and localization of the Ces5a gene in the testes of three litters of rats at different postnatal (PN) days. RESULTS: The expression of Ces5a mRNA was found in the testis tissue of the rats at 2－65 PN days, low at 2－12 days, decreased to the lowest level at 14－16 days (P < 0.05), but significantly increased at 20－35 days (P < 0.05), and elevated to the highest level at 40－65 days (P < 0.05). The expression of the Ces5a protein was also observed in the testis tissue of the rats at 2－65 PN, low at 2－12 days, with no significant change at 14－16 days (P > 0.05), but markedly increased at 20－35 days (P < 0.05), and again with no significant change at 40－65 days (P > 0.05). The Ces5a protein was expressed in the spermatogonia, spermatocytes and round sperm cells. CONCLUSIONS: The Ces5a gene may be involved in the proliferation and meiosis of rat spermatogonia and play a special role in round spermatogenesis and sperm deformation.

[Expression of the MYL2 gene in the development of rat testis tissue].

OBJECTIVE: To study the expression of the gene of myosin regulatory light chain-2 (MYL2) in the development of rat testis tissue. METHODS: Using real-time PCR and immunohistochemistry, we determined the mRNA transcription level and protein expression of MYL2 in the rat testis. RESULTS: The mRNA expression of the MYL2 gene changed in an age-dependent manner, reaching the highest value on postnatal day (PND) 2, then dropped rapidly till PND 8, increased slowly on PNDs 10 and 12, decreased on PND 14, rose slightly from PND 15 and rapidly on PNDs 20 and 25, and declined slowly from PND 65. Immunohistochemistry showed that the MYL2 protein was mainly expressed in testicular sperm cells. CONCLUSIONS: The MYL2 gene may be involved in the proliferation of spermatogonial stem cells and the process of sperm cells developing into mature sperm.

Tribbles homolog 3 expression in spermatogonial stem cells of rat testes.

Tribbles are a family of signal-regulating proteins shown to coordinate the action and the suppression of different pathways. Tribbles homolog 3 (Trib3), the best-studied member of the mammalian tribble family, has a key function in determining cell fate when responding to environmental challenges. Trib3 effects are also modulated by its direct interaction with other signaling molecules. We found that Trib3 is highly expressed in the early development of rat testis, at just the time when the gonocytes resume proliferation to give rise to A spermatogonia. Immunofluorescence staining of cross-sections of rat testis and cultured spermatogonial stem cells (SSCs) also confirmed that Trib3 is expressed in rat SSCs.

The proliferation and differentiation of spermatogonial stem cells in the frist wave of spermatogenesis in rats with Trib3 gene knockout.

This study explores the effects of Trib3 gene knockout on adult male rat spermatogenesis. Using CRISPR/Cas9, we knocked out the Trib3 gene in Wistar rats. Results indicate altered expression of PLZF, ID4, and c-KIT in knockout rats, suggesting impaired spermatogonial stem cell proliferation and differentiation. Histological analysis reveals reduced seminiferous tubule area and decreased spermatocyte numbers. Mating experiments demonstrate reduced offspring rates after the second self-mating in knockout rats. SYCP3, a meiosis marker, shows elevated expression in knockout rat testes at 14 days postpartum, suggesting an impact on reproductive processes. ELISA results indicate decreased testosterone, FSH, and FGF9 levels in knockout rat testicular tissues. In conclusion, Trib3 gene deletion may impede spermatogonial self-renewal and promote differentiation through the FSH-FGF9- c-KIT interaction and p38MAPK pathway, affecting reproductive capacity. These findings contribute to understanding the molecular mechanisms regulating spermatogenesis.

Transcriptome Profiling of Developing Testes and First Wave of Spermatogenesis in the Rat.

Spermatogenesis is a complicated course of several rigorous restrained steps that spermatogonial stem cells undergo to develop into highly specialized spermatozoa; however, specific genes and signal pathways, which regulate the amplification, differentiation and maturation of these cells, remain unclear. We performed bioinformatics analyses to investigate the dynamic changes of the gene expression patterns at three time points in the course of the first wave of rat spermatogenesis. Differently expressed genes (DEGs) were identified, and the features of DEGs were further analyzed with GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes) and Short Time-series Expression Miner (STEM). A total of 2954 differentially expressed genes were identified. By using STEM, the top 10 key genes were selected in the profile according to the enrichment results, and the distinguishable biological functions encoded by these DEGs were automatically divided into three parts. Genes from 6, 8 and 10 days were related to biosynthesis, immune response and cell junction, and genes from 14, 15 and 16 days were related to energy metabolic pathways. The results also suggest that genes from 29, 31 and 35 days may shift metabolic to sperm motility, sperm flagellum and cilium movement.

Effects of exogenous melatonin on expressional differences of immune-related genes in cashmere goats.

The interplay between melatonin and immune system is well recognized in humans. The true integration of research on cashmere goat is still far from clear, especially for cashmere goat maintained in wool and cashmere growth. In this study, we applied various approaches to identify the complex regulated network between the immune-related genes and transcription factors (TFs) and to explore the relationship between melatonin and gene expression in cashmere goats. In total, 1,599 and 1756 immune-related genes were found in the blood and skin of cashmere goats, respectively, and 24 differentially expressed immune-related GO terms were highly expressed in blood after melatonin implantation. We studied the melatonin-dependent networks between the TFs and immune-related genes in cashmere goat. The 3 major regulatory networks were interconnected through TFs. The TFs, such as PHF5A, REXO4, STRAP, JUNB, GATAD2A, ZNF710, and VDR, were also expressed in the blood and skin tissue of cashmere goat. In addition, most genes in these networks, such as VDR, JUNB, and Trib3, were involved in WNT pathway, which is related to cashmere wool growth regulation. On the network basis, we developed a knockout mouse model to identify the network interaction. We observed that 8 high-sulfur protein genes, 12 keratin (KRT) genes, and 19 keratin associated protein (KRTAP) genes related to the growth of cashmere wool were almost not expressed in Trib3 -/- rat skin. Our results suggested that the expression of genes related to wool and cashmere growth may be regulated by the interaction network between genes affected by melatonin and immune-related genes. In summary, we outlined some particularly promising ways for future research on immune-related genes of cashmere goats and the role of melatonin in wool and cashmere growth.

[Comparative study of two high-efficiency methods for purifying spermatogonial stem cells].

Objective: To compare the advantages and disadvantages of the differential attachment method and immunomagnetic bead method for purification of mouse spermatogonial stem cells (mSSCs). Methods: Ten male C57BL/6 mice aged 12～15 days were selected and sacrificed by cervical dislocation. Testes were collected and the seminiferous tubule single cell suspension was obtained by enzymatic digestion. mSSCs were purified by using the differential attachment method and immunomagnetic bead method respectively. Then a detailed comparison of the two methods in terms of cell number, separation efficiency, and impact on cell proliferation and growth was conducted. Results: Both of the methods could isolate and purify stem cells from single cell suspension of mouse seminiferous tubules. mSSCs showed typical grape cluster-like clones in vitro culture, which could be continuously cultured and proliferated for over 3 months in vitro. The testes of 10 mice could obtain 3×105±0.4×105 mSSCs (n=5) by differential attachment method, cell recovery rate (the number of cells after purification/the number of cells of the single cell suspension of seminiferous tubules) was 1.5%±0.1%; 6×105±0.4×105 mSSCs (n= 5) could be obtained by immunomagnetic bead method. The recovery rate was about 3%±0.1%, and the number of stem cells obtained by the immunomagnetic bead method was higher. The stem cells obtained by the differential attachment method were more pure, because the stem cell colonies were preferentially obtained after 5 days of in vitro culture, while the stem cells obtained by the immunomagnetic bead method needed to be cultured for about 10 days before the obvious cell colonies could be observed, but the two types of purification method had no obvious effect on the long-term growth of cells in vitro. Conclusion: Both methods can get high quality mSSCs, but both methods have their own advantages and disadvantages. The differential attachment method is more economical and practical than the other, it does not require special equipment, but the stem cell number obtained is relatively lower and the time needed is longer.

[Effect of dopamine receptor agonist apomorphine on scopolamine induced memory deficits in mice].

OBJECTIVE: To research the mechanism of dopamine (DA) controlled memory in mice. METHODS: Mice received i.p. injection of scopolamine (0.3 mg/kg, SCOP 0.3, and 3.0mg/kg, SCOP 3.0, respectively, n = 10) and saline (NS, n = 10) for 60 days in experiment 1. Memory of mice was detected by dark avoidance behavior in the 53" d and the 60"' d. Animals were sacrificed after the memory test; brain tissues were processed for Fos-ir and TH-ir by immunohistochemistry. Mice were divided into four groups according results of expri-ment 1, they received i.p. injection of apomorphine (0.1 mg/kg, APO 0.1, 0.5 mg/kg, APO 0.5, and 2.0 mg/kg, APO2.0 respectively, n = 10). RESULTS: Memory was inhibited in mice injected scopolamine 3.0 mg/kg. Latency was significantly less than in NS group, only 1/ 4 that of NS group (P > 0.05). The number of mistake of SCOP 3.0 group increased about four times than that of NS group (P > 0.05). But there was no difference of latency and number of mistake between SCOP 0.3 and NS group in expriment 1. Scopolamine-induced memory deficit was associated with decreased cellular activation, indicated by Fos immunoreactive (ir) staining, in NAcc CA1 and CA3 (P < 0.05), and also associated with decreases in the number of cells labeled for tyrosine hydroxylase (TH-ir), the rate limiting enzyme for dopamine conversion (P < 0.01) and the number of cells co-labeled for TH-ir/Fos-ir (P <0.01) in the ventral tegmental area(VTA), apomorphine lessened scopolamine-induced memory deficit in experiment 2. The number of cells co-labeled for TH-ir/Fos-ir (P <, 0.05) was increased in VTA after apomorphine treatment. CONCLUSION: Apomorphine lessened scopolamine-induced memory deficit in mice by increasing DA activities in VTA.

Inhibition of mammalian target of rapamycin signaling by CCI-779 (temsirolimus) induces growth inhibition and cell cycle arrest in Cashmere goat fetal fibroblasts (Capra hircus).

The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase. It plays an evolutionarily conserved role in regulating cell growth, proliferation, survival, and metabolism via different cellular processes. The purpose of this study was to explore the inhibitory effects of CCI-779 (temsirolimus), a specific mTOR inhibitor, on mTOR signaling, and examine the mechanism of cell growth suppression by CCI-779 in Cashmere goat fetal fibroblasts (GFb cells). GFb cells were sensitive to CCI-779 and the survival rate of cells treated with >3.0 µM of CCI-779 was significantly reduced compared with the control (p<0.01). CCI-779 inhibited the phosphorylation of mTOR (at Ser2448) and S6 (at Ser240/244), and the expression of mTOR, p70S6K, and S6. Thus, CCI-779 was toxic to GFb cells, and it induced a dose-dependent decrease in cell proliferation and caused G1/S cell cycle arrest. Taken together, these data show that CCI-779 can inhibit mTOR signaling and proliferation in GFb cells in vitro. Therefore, mTOR is an important regulator for GFb cell growth and proliferation.

E-cadherin can be expressed by a small population of rat undifferentiated spermatogonia in vivo and in vitro.

Spermatogonial stem cells (SSCs) maintain gamete production in the testes throughout adult life by balancing self-renewal and differentiation. In vitro culture of SSCs is a crucial technique for gene manipulation of SSCs to generate transgenic animals, for transplantation of SSCs to restore male fertility for infertile man, and for generation of pluripotent stem cells from SSCs to differentiate into various cell lineages. Isolation of highly purified SSCs is an all-important component for development of these techniques. However, definitive markers for SSCs, which purify SSCs (100% enrichment), are unknown. SSCs of many species can colonize the mouse testis; thus, we reasoned that same molecules of SSCs are conserved between species. In mouse, undifferentiated spermatogonia express the surface marker E-cadherin. The hypothesis tested in this work was that E-cadherin (also known as CDH1) can be expressed by undifferentiated spermatogonia of rat testes. In this paper, cross-section immunohistochemistry and whole-mount immunohistochemistry of rat seminiferous tubules were conducted to show that E-cadherin-positive cells were small in number and there are single, paired, and aligned spermatogonia attached along the basement membrane. During in vitro culture period, the undifferentiated rat spermatogonial colonies co-expressed E-cadherin and glial-derived neurotrophic factor family receptor alpha-1 or E-cadherin and promyelocytic leukemia zinc finger. Data collected during the study demonstrate that E-cadherin is expressed by a small population of rat undifferentiated spermatogonia both in vivo and during in vitro culture period.