Comparative Expression Analysis of Spermatogonial Stem Cell Markers in Cattle and Sheep

Iranian Journal of Applied Animal Science

دوره 13، شماره 4، اسفند 2023، صفحه 685-693   

نوع مقاله: Research Articles

نویسندگان

F. Nasri Ahangar¹؛ M. Zandi^{* 1}؛ M.R. Sanjabi¹؛ A. Ghaedrahmati²

¹Department of Agriculture, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran

²Department of Animal Science, Agricultural Science and Natural Resources University of Khuzestan, Mollasani, Khuzestan, Iran

چکیده

Spermatogenesis is supported by stem cells called spermatogonial stem cells (SSCs), which are capable of transmitting information to the next generation. However, little is known on the specific markers of SSCs in farm animals. We investigated the expression of cdh1, cmyc, bcl6b, plzf, gfra1, nanog, vasa, thy1 and uchl1, as specific markers of SSCs, in bovine and ovine SSCs. The expression of the studied genes was conducted by real-time polymerase chain reaction method. For this reason, the enzymatic digestion process for two times was used to achieve SSCs from male calves and ram lambs testes. Then, filtration and differential plating techniques increased the SSC number in the cell suspension caused by mechanical and enzymatic digestions. Sertoli cells treated with Mitomycin-C were applied to obtain the feeder layer. Culturing of the stem cells was done on Sertoli cell feeder layer. Our results revealed that the expression of nanog and plzf was similar in bovine and ovine SSCs. Unlike bovine SSC colonies, cdh1 gene was not expressed in colony of ovine SSCs and its use as a specific marker of sheep SSCs is not suggested. The expression of uchl, vasa, thy1 and cdh1 genes was significantly higher in bovine SSCs and the bcl6b and cmyc expression was significantly higher in ovine SSCs compared to each other (P<0.05). From the results of this study nanog, plzf, uchl1, vasa, thy1 and cdh1 are suggested as markers of bovine and nanog, plzf, bcl6b and cmyc genes as markers of ovine SSCs.

کلیدواژه‌ها

cell specific Factor؛ molecular markers؛ pluripotency؛ surface markers؛ transcrip-tion factor

اصل مقاله

INTRODUCTION

SSCs can be found on the basement membrane of the seminiferous tubules in testes and can differentiate and self-renew into spermatogenic cells at various developmental phases (Wu et al. 2022). SSCs are needed to be recognized and selectively purified before germ cell culture or transplant; however, they are not easy due to no available specific markers as well as the limited generation of them in the testis (Herrid et al. 2007). Moreover, due to limited information regarding surface markers in ovine SSCs, other techniques have been prevented, like fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) for SSC enrichment (Borjigin et al. 2010). SSCs self-renewal is controlled by many key players (Cai et al. 2020). For instance, promyelocytic leukemia zinc finger protein (PLZF) or ZBTB16 is a sheep undifferentiated spermatogonia (Zhang et al. 2014). PLZF as a transcription factor is necessary for the self-renewal and maintenance of SSCs and can be expressed by mouse undifferentiated spermatogonia. It is also expressed in a sub-population of gonocytes and SSCs/progenitors in cattle, pigs, goats, sheep, non-human primates and equids (Buaas et al. 2004; McMillan et al. 2014; Zheng et al. 2014a). It was used to assess the number of the putative undifferentiated spermatogonia. However, the cells that express PLZF is not separable alive using immunologic methods because of the localization of the marker in the cell nucleus (Zhang et al. 2014). Molecular markers that are expressed exclusively in male germ line stem cells (MGSCs) are needed to be identified, isolated and purified. Different molecular markers are used to recognize and assess MGSCs. PLZF expression by MGSCs has been reported in pigs, rodents, bulls, sheep, humans and nonhuman primates (Zheng et al. 2014b).

 

MATERIALS AND METHODS

Chemicals and antibodies

Unless otherwise specified, the chemical agents were purchased from Sigma (USA).

 

Samples preparation

Testes of a healthy male calves and ram lambs were collected quickly following slaughter and transferred to lab within 2-3 hours. In brief, the tunica albuginea was removed for the first enzymatic digestion. 6-10 g testis tissue was rinsed in deionized water 3 times and normal physiological saline 10 times then sprayed to 70% ethanol. When the scrotum and tunica albuginae were removed, phosphate buffer saline was used to wash testes, followed by cutting the testes to small pieces. Using a two-step enzymatic isolation procedure, individual testicular cells were obtained according to Izadyar et al. (2002) with some changes. A segment of 2 g tissue was transferred into a 15-mL Corning™ falcon 50 mL conical centrifuge tubes. Dissection of the testis tissue free of the connective tissue and rete testis was done and placed in a Petri dish containing 5 mL DMEM with 50 µg/mL gentamycin was filter by 22/5 µm filter. The tissue was washed 4 times with 5 mL DMEM with 50 µg/mL gentamycin. The pellet was transferred into a 50-mL tube.

 

Enzymatic isolation and culture of SSCs

First step enzymatic digestion, was carried out with 1 mg/mL hyaluronidase type II,1 mg/mL trypsin (Inoclon), 5 μg/mL DNase and 1 mg/mL collagenase. Incubation was done in a shaker incubator (200 cycles/min) (37 ˚C for 45 min). The collected dispersed tissue was collected by centrifugation (1000 rpm for 2 min). The supernatant was removed from the plate. Considering the second enzymatic digestion, the pellet suspension was prepared in DMEM, including 1 mg/mL collagenase, 1 mg/mL hyaluronidase type II, and 5 μg/mL DNase. To achieve favorite cell population, cellular suspension underwent centrifugation (1000 rpm for 2 min). The digestion was discontinued by adding 3 mL DMEM of 10% fetal bovine serum (FBS).

 

Enrichment of SSCs

Filtration of the supernatant was done through 80-µm and then 60-µm nylon net filters for SSC enrichment. The SSCs was seeded onto lectin- bovine serum albumin (BSA) coated 60 mm culture dishes according to Jafarnejad et al. (2018). To prepare the lectin-BSA coated dishes 5 µg/mL lectin from Datura stramonium agglutinin was dissolved in DPBS and washing them by addition of 0.6% BSA in DPBS at room temperature for 2 h. After that, the dishes were rinsed using BSA and kept at room temperature for 2 hours. They were subjected to coating BSA at room temperature for another 2 h. The cultures or cells on the lectin-coated dishes underwent incubation in a CO₂ incubator (5-6 h at 37 ˚C). Using the incubation process, most of the contaminated cells can bind to lectin-BSA. The suspension was transferred to 15 mL tube, and it seemed to contain SSCs. It was predicted that the medium subsequently centrifuged at 1000 rpm for 5 min, contains SSCs and the pellet re-suspension in DMEM.

 

Preparing the feeder layers

The Sertoli cell feeder layer was prepared as described in Jafarnejad et al. (2018) study with some modifications. In summary, the rest of attached cells in the dishes coated with lectin-BSA from enrichment of SSCs step were cultured at 37 ˚C in a humidified room using 5% CO₂ for two to three days. Incubation was done for growing Sertoli cells, then they were treated with trypsin-EDTA (0/25%) and used for sub-culturing a cell culture flask (50 mL) for propagation. Then, the Sertoli cells received mitomycin-C (10 g/mL) for 3 hours for inactivation. Finally, were rinsed five times in DPBS and DMEM supplemented with 10% FBS to remove any remaining mitomycin C, and were used as feeder cells for SSC culture.

 

Culture and characterization of SSCs

Culturing of the isolated SSCs was done on the Sertoli cells feeder layer coated culture flasks containing DMEM medium treated with 10 μg/mL GDNF, 10% FBS, penicillin (100 IU/mL) and streptomycin (50 mg/mL) and then incubation was done in an incubator using 5% CO₂ in air at 37 ˚C. Replacement of the culture medium was done every third day. SSC colonies were found following ten days and alkaline phosphatase (AP) staining was performed to characterize them. For this, washing of SSC colonies was done twice using DPBS followed by staining by an AP kit (Sigma, Catalogue No. 86 C) as instructed.

 

RNA isolation and reverse transcription

Trizol reagent (Invitrogen, USA) was applied to extract total RNA as instructed. Its quantity and quality was assessed by NanoDrop spectrophotometer (Dynamica Scientific Ltd., United Kingdom) by measuring the absorbance at 260 nm, and then treatment with DNAse (Ambion Inc., Houston, Texas, USA) to remove probable contaminating genomic DNA. For each specimen, total RNA (0.5 mg) was applied for the synthesis of first-strand complementary DNA (cDNA). MMLV enzyme and oligo dT primers (Takara, Japan) was used for reverse transcription. The cDNAs were used to measure RNA abundance using quantitative real-time PCR.

 

Real-time PCR

The PCR reaction was fixed in a final volume of 10 µL, consisting of 0.8 µL of each primer (0.2 µM) (forward and reverse), 10 μL with 1.4 µL nuclease-free water, 5 µL Syber Green, and 2 µL template. To activate the polymerase, the thermal cycling conditions including initial denaturation (94 ˚C/15 minutes), 40 amplification cycles of denaturation (95 ˚C/10 second) and annealing specific primers based on Table 1 for 15 sec and 72 ˚C for extension for 20sec. The reactions ended by a final extension (72 ˚C/5 minutes). Also, β-actin gene was applied as the internal control to investigate the expression levels of the gene mRNA and was calculated by the (∆∆CT) method. The used primers in are mentioned in Table 1.

 

Table 1 The used primers sequences (R, reverse primer; F, forward primer)

 

Figure 1 Alkaline phosphatase staining was used to identify SSCs. Alkaline phosphatase expressed in both of bovine (A) and ovine SSCs (B) 10 days after culture (Bar=0.5 mm)

 

Fold change of gene expression was calculated as a ratio of expression levels of treated groups to the expression level of the control group (Livak and Schmittgen, 2001).

 

Statistical analysis

The experimental procedures were repeated at least three times. Data analysis was done using t-test to compare two means (SPSS, 2011). The results have arrived as the Mean ± Standard Error of Mean (SEM) and statistical significance was fixed at (P<0.05).

 

RESULTS AND DISCUSSION

The bovine and ovine SSCs suspension was achieved by two times enzymatic digestion. SSCs formed colonies seven days following culture and attachments among colonies could be observed at 10^th day. They were positive for alkaline phosphatase staining Figure 1. Real-time PCR was done in the removed SSCs to assess the expression of a subset of pluripotency markers, and also germ cell-specific genes. Markers used for SSC characterization in this study included: cdh1, cmyc, bcl6b, plzf, gfra1, nanog, vasa, thy1 and uchl1. β-actin is considered as housekeeping gene. Results demonstrated that the expression of nanog and plzf was similar in bovine and ovine SSCs Figures 2-3. The expression of uchl1, vasa, thy1 and cdh1 genes was significantly higher in bovine SSCs in comparison with ovine SSCs Figures 4-7. The expression of vasa in ovine SSCs was very low, and thy1 and cdh1 was close to zero. The expression of bcl6b, cmyc and gfra1 genes was higher in sheep than in cattle, so this difference was significant in bcl6b and cmyc genes (P<0.05) Figures 8-10. Use of several methods and markers for recognition and enrichment of SSCs is important because of the low number of SSCs in testes (Abbasi et al. 2013). In comparison to other species, such as rodents, there are not many known spermatogonia phenotypic markers in domestic animals. However, some markers can be consistently expressed in spermatogonia from domestic species (Zheng et al. 2014a). In this study expression of cdh1, cmyc, bcl6b, plzf, gfra1, nanog, vasa, thy1 and uchl1 genes were studied in bovine and ovine SSCs. From the genes under study the expression of pluripotency gene nanog and germ cell-specific factor plzf was similar in bovine and ovine SSCs. Bovine gonocytes could express NANOG in the neonatal testis until the migration of gonocytes to the basal membrane (Fujihara et al. 2011). The similar NANOG expression patterns in various species such as pig (Goel et al. 2008), marmoset (Mitchell et al. 2008), and human (Hoei Hansen et al. 2005) indicate the highly conserved NANOG role in spermatogenesis in mammals (Fujihara et al. 2011). According to Borjigin et al. (2010) PLZF-positive cells in ovine testis are a Type A spermatogonia subpopulation representing the undifferentiated spermatogonia, such as SSCs that is self-renewed. Also, plzf identified Type A spermatogonia in the bovine testis and are expressed by a bovine spermatogonia subpopulation, and possibly limited to SSCs and very early spermatogonia (McMillan et al. 2014). PLZF as a sequence-specific DNA-binding protein is able to repress the kit transcription, a spermatogonial differentiation hallmark leading to maintaining the source of SSCs in mice. For the first time Bahadorani et al. (2011) reported expressing PLZF in undifferentiated spermatogonia in sheep and goats, evidenced by positive reaction with the anti-PLZF polyclonal antibody. Thus, because positive PLZF-stained cells were merely found at the base of seminiferous tubules, PLZF is capable of expressing in undifferentiated spermatogonia.

 

Figure 2 Comparison of nanog gene expression levels between bovine and ovine SSCs. Relative fold change determined by quantitative real-time PCR analysis. The data were normalized with β-actin expression and given as relative to the control (bovine SSCs)

 

Figure 3 Comparison of plzf gene expression levels between bovine and ovine SSCs. Relative fold change determined by quantitative real-time PCR analysis. The data were normalized with β-actin expression and given as relative to the control (bovine SSCs)

 

Figure 4 Comparison of uchl1gene expression levels between bovine and ovine SSCs. Relative fold change determined by quantitative real-time PCR analysis. The data were normalized with β-actin expression and given as relative to the control (bovine SSCs)

 

Figure 5 Comparison of vasa gene expression levels between bovine and ovine SSCs. Relative fold change determined by quantitative real-time PCR analysis. The data were normalized with β-actin expression and given as relative to the control (bovine SSCs)

 

Figure 6 Comparison of thy1gene expression levels between bovine and ovine SSCs. Relative fold change determined by quantitative real-time PCR analysis. The data were normalized with β-actin expression and given as relative to the control (bovine SSCs)

 

Figure 7 Comparison of cdh1gene expression levels between bovine and ovine SSCs. Relative fold change determined by quantitative real-time PCR analysis. The data were normalized with β-actin expression and given as relative to the control (bovine SSCs)

 

Figure 8 Comparison of bcl6b gene expression levels between bovine and ovine SSCs. Relative fold change determined by quantitative real-time PCR analysis. The data were normalized with β-actin expression and given as relative to the control (bovine SSCs)

 

Figure 9 Comparison of c-myc gene expression levels between bovine and ovine SSCs. Relative fold change determined by quantitative real-time PCR analysis. The data were normalized with β-actin expression and given as relative to the control (bovine SSCs)

 

Figure 10 Comparison of gfral gene expression levels between bovine and ovine SSCs. Relative fold change determined by quantitative real-time PCR analysis. The data were normalized with β-actin expression and given as relative to the control (bovine SSCs)

 

Our results showed that cdh1 gene only expressed in bovine SSCs and uchl1, vasa and thy1 genes were more expressed in bovine SSCs in compare to ovine SSCs. Inconsistent results are available on the CDH1 expression and the localization of CDH1-positive cells in testes of rodents. CDH1 was not observed in the testis, whereas many recent papers have declared opposite results. Tokuda et al. (2007) reported CDH1 expression in some germ cells in the testes of mice. There were limited number of CDH1-positive cells in the adult mouse testis, indicating the reason why CDH1 staining has been missed in some studies. In contrast to our results in ovine SSCs, Zhang et al. (2014) found that CDH1 was expressed in undifferentiated spermatogonia of sheep by immune histochemistry examination of frozen section of seminiferous tubules of sheep testis. Yu et al. (2014) reported CDH1 expression by some spermatogonia, and CDH1 localization was limited to the single and paired spermatogonia basement membrane. Zheng et al. (2014a) and others reported the UCHL1 expression in pre-pubertal testes of buffalo, pigs, cattle, sheep and, goats (Abbasi et al. 2013; Yu et al. 2014). UCHL1 expression was observed in pre-meiotic male germ cells and showed an affinity for somatic cells, making it a good marker for spermatogonia in domestic testes (Zheng et al. 2014a). VASA expression was observed in bovine gonocytes in the neonatal testes and its expression continued in spermatogonia until migration to the basement membrane (Fujihara et al. 2011). VASA expression was detected in the adult bovine testis, in differentiated germ cells, like round spermatids and spermatocytes, but not in spermatogonia. The same VASA expression patterns were reported in mouse (Toyooka et al. 2000), human (Castrillon et al. 2000), and pig (Lee et al. 2005) adult testes. Accordingly, besides mice, cattle, and rats, VASA antibody is applicable to recognize germ cells in goats and sheep (Bahadorani et al. 2011). In agreement with results of Bahadorani et al. (2011), the VASA protein belonging to the ATP-dependent RNA helicase of the DEAD-box family protein, has a conserved structure; thus, the PCR was applied to verify this point across many species from Caenorhabditis elegans to humans (Zeeman et al. 2002). In contrast to PLZF and VASA as respectively cytoplasmic and nuclear markers, THY1 has been reported as a surface marker. THY1 is a GPI (N-glycosylated, glycophosphatidyl inositol; 25-37 kDa) anchored protein that was initially detected as a thymocyte antigen and was found in some stem cell sources, such as SSCs (Rege and Hagood, 2006). THY1 expression as a conserved surface marker of undifferentiated spermatogonia is a common phenotype of SSCs in many mammals and showed conservation in other livestock (Reding et al. 2010). Our results indicated that the bcl6b, cmyc and gfra1genes were more expressed in ovine SSCs. BCL6B expression was upregulated by GDNF and suppressed in the AKT presence (Protein Kinase B) inhibitor (Yu et al. 2014). Oatley et al. (2006) declared that BCL6B has a significant role in SSCs maintenance in vitro, but it is a crucial transcription factor for SSCs proliferation self-renewal, and survival. c-myc gene as a pluripotency marker, code for important transcription factors in self-renewal division and the cell cycling of many stem cell types (Bojnordi et al. 2017). Yamanaka’s transcription factors, including c-Myc (OSKM factors), Oct3/4, Klf4 and Sox2 are associated with the somatic differentiated cell reprogramming into induced pluripotent stem cells (iPSCs) (Aoi et al. 2008). The KLF4 and c-Myc showed higher expression levels in SSCs than ES cells (Corbineau et al. 2017). GDNF maintained goat SSC self-renewal and GDNF could up-regulate c-Myc expression by the PI3K/Akt pathway to increase SSC proliferation in goats (Niu et al. 2016). Gfra1 is a surface marker for undifferentiated spermatogonia in the testes of mice (Buageaw et al. 2005), and can be expressed in a sub-population of gonocytes in porcine testes of neonates (Lee et al. 2013).

 

CONCLUSION

Our results revealed that the expression of nanog and plzf was similar in bovine and ovine SSCs. Unlike bovine SSC colonies, cdh1 gene was not expressed in colony of ovine SSCs and its use as a specific marker of sheep SSCs is not suggested. The reason for the lack of expression of cdh1 gene in ovine SSCs can be the difference in the expression of this gene in single and paired cells in comparison with the colony stage. The results of the present study showed that uchl1, vasa, thy1, and cdh1 genes and bcl6b and cmyc genes were more expressed in bovine and ovine SSCs, respectively.

 

ACKNOWLEDGEMENT

The authors are grateful to Agricultural Institute of IROST for providing this project with laboratory facilities and other technical support.

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