High Temperature Inhibits The Differentiation of Spermatogonial Stem Cells


 Background

As one of the factors of male infertility, high temperature induces apoptosis of differentiated spermatogenic cells, sperm DNA oxidative damage, and changes in morphology and function of Sertoli cells. Spermatogonial stem cells (SSCs) is a kind of germline stem cells which maintain the spermatogenesis through self-renewal and differentiation. At present, however, the effect of high temperature on SSC differentiation in vitro has not been reported.
Methods

In the present study, we used in vitro differentiation model of SSCs to research the effect of heat stress treatment on SSC differentiation. Firstly, real-time PCR was used to detect the expression levels of self-renewal and differentiation marker genes in differentiation-cultured SSCs after heat stress treatment. Then, the effect of heat stress on the transcriptome of differentiation cultured SSCs was analyzed by RNA-seq. Enrichment of functions and signaling pathways analysis were performed based on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
Results

We found that 2 days of 37℃ or 43℃ (30min/d) heat stress treatment significantly inhibited SSC differentiation. The differentiation related genes c-kit, stra8, Rec8, Sycp3 and Ovol1 were down-regulated after 2 days and 4 days of heat stress at 37℃. The transcriptome of SSCs was significantly differentially expressed on the second day and fourth day after heat stress treatment at 37℃. In total, 1660 and 7252 differentially expressed genes (DEGs) were identified by RNA-Seq in SSCs treated with heat stress at 37℃ for 2 days and 4 days respectively, compared with those cultured at 34℃. KEGG pathway analysis showed that P53, ribosome and carbon metabolism signaling pathways promoting stem cell differentiation were significantly enriched after heat stress treatment at 37℃.
Conclusion

These results indicate that high temperature at 37℃ significantly inhibits SSC differentiation and promotes enrichment of P53, ribosome and carbon metabolism signaling pathways in stem cell differentiation, providing a reference for the pathogenesis of heat-induced azoospermia.


Abstract Background
As one of the factors of male infertility, high temperature induces apoptosis of differentiated spermatogenic cells, sperm DNA oxidative damage, and changes in morphology and function of Sertoli cells. Spermatogonial stem cells (SSCs) is a kind of germline stem cells which maintain the spermatogenesis through self-renewal and differentiation. At present, however, the effect of high temperature on SSC differentiation in vitro has not been reported.

Methods
In the present study, we used in vitro differentiation model of SSCs to research the effect of heat stress treatment on SSC differentiation. Firstly, real-time PCR was used to detect the expression levels of self-renewal and differentiation marker genes in differentiation-cultured SSCs after heat stress treatment. Then, the effect of heat stress on the transcriptome of differentiation cultured SSCs was analyzed by RNA-seq. Enrichment of functions and signaling pathways analysis were performed based on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Results
We found that 2 days of 37℃ or 43℃ (30min/d) heat stress treatment signi cantly inhibited SSC differentiation. The differentiation related genes c-kit, stra8, Rec8, Sycp3 and Ovol1 were down-regulated after 2 days and 4 days of heat stress at 37℃. The transcriptome of SSCs was signi cantly differentially expressed on the second day and fourth day after heat stress treatment at 37℃. In total, 1660 and 7252 differentially expressed genes (DEGs) were identi ed by RNA-Seq in SSCs treated with heat stress at 37℃ for 2 days and 4 days respectively, compared with those cultured at 34℃. KEGG pathway analysis showed that P53, ribosome and carbon metabolism signaling pathways promoting stem cell differentiation were signi cantly enriched after heat stress treatment at 37℃.

Conclusion
These results indicate that high temperature at 37℃ signi cantly inhibits SSC differentiation and promotes enrichment of P53, ribosome and carbon metabolism signaling pathways in stem cell differentiation, providing a reference for the pathogenesis of heat-induced azoospermia.

Background
The World Health Organization predicts that infertility will become the third most intractable disease after cancer and cardio-cerebrovascular disease in the 21st century [1]. Infertility occurs in 10-15% couples of childbearing age, and male factors account for 50% of cases [2]. A Global Burden of Disease survey reported that from1990 to 2017, the agestandardised prevalence of infertility increased annually by 0.291% in men [3]. Heat is one of the causes of male infertility [4]. Cryptorchidism or increased scrotal temperature leads to non-obstructive azoospermia or asthenospermia.
SSCs are the source of spermatogenesis and their differentiation is tightly regulated. SSCs are widely considered to be single undifferentiated spermatogonia cells existing on the basement membrane in seminiferous tubules. SSCs belong to A singal spermatogonia (A s ). In rodents, A s spermatogonia generate two A s spermatogonia without an intercellular bridge. Subsequent cell divisions of the A pr spermatogonia generate A aligned−4 , A aligned−8 , and A aligned−16 (A al ), which will differentiate to type A 1 spermatogonia. The A s , A pr , and A al spermatogonia are called undifferentiated spermatogonia (A undiff ), retaining the potential to differentiate into A 1 , A 2 , A 3 , A 4 , In, and B spermatogonia, which goes into meiosis to form primary spermatocyte, secondary spermatocyte, and eventually form sperm [5,6]. Retinoic acid (RA) is the inducer of differentiation in SSCs and induces differentiating spermatogonia to express early markers of spermatogenic differentiation, including KIT and STRA8 [7,8]. To become sensitive to the differentiation-inducing stimulus (RA), A undiff need to exit the self-renewing state and undergo differentiation priming [8,9]. This transition involves the activation of the mTORC1 pathway that plays a critical role in maintenance of SSCs, and aberrant mTORC1 activation promotes stem cell exhaustion [10][11][12][13]. WNT/β-catenin signaling plays an important role in the differentiation priming of A undiff by promoting the transition from self-renewing to RA-responsive state [14,15]. Interestingly, Tokue et al identi ed SHISA6 as a novel marker for a speci c subset of GFRα1-expressing A undiff [16]. SHISA6 is suggested to act as a WNT signaling inhibitor and thus confer resistance to the differentiation-priming program. Mir-322 regulates SSC differentiation through the WNT/β-catenin signaling pathway, and miR-322 overexpression decreased STRA8, C-KIT and BCL6 expression [17]. Luo et al. found that SSCs can be derived into ovarian organoids and produce offspring [18]. In the study, they transdifferentiated SSCs into oocytes by transduction of H19, Stella, and Zfp57 and inactivation of Plzf in SSCs. The regulation of SSC differentiation process is very complex, and any failure of the regulation can lead to male infertility. The causes of male infertility are wide ranging and poorly understood in most cases [19][20][21].
Temperature is considered to be a key regulator of reproductive activity and testicular homeostasis. Spermatogenesis and sperm maturation are closely related to temperature and require a temperature slightly lower than the normal body temperature 2-7℃ [22,23]. Severe or repetitive heat exposures often induce male subfertility or infertility due to reduced sperm output and qualities [24]. Therefore, high temperature is an important external factor affecting multiple stages of spermatogenesis and sperm function. In the heat stress condition, mammalian male germ cells show a variety of changes in cellular events including stress granule (SG) formation, DNA damage and apoptosis [25]. A study showed that adult male mice were exposed to an elevated ambient temperature of 35 for 24h and followed by recovery 1 day, identi ed elevated sperm mitochondrial ROS generation, increased sperm membrane uidity, pachytene spermatocytes and round spermatids DNA damage [26]. The most relevant consequence of heat stress on the testis is death of germ cells via apoptosis. Previous research indicate that the testicular germ-cell loss by apoptosis after exposure to abdominal heat stress occurs [27]. Studies have shown that p38 MAPK pathway regulates both apoptosis and spermatocyte differentiation [28][29][30].
However, the role and mechanism of heat stress in regulating SSC development is unclear, due to the small number of SSCs in the mouse testis (only 0.02-0.03% of total testis cells ) [31]. Our previous study shown that heat shock treatment at 43℃ for 45 min signi cantly inhibited SSC self-renewal through S phase cell cycle arrest but not apoptosis [32]. But there are few reports on the effect of high temperature on the differentiation of SSCs. In this study, we rst successful used in vitro differentiation model of SSCs studied the effect of heat stress treatment on SSC differentiation.
Heat stress treatment of differentiated culture SSCs The SSCs used in differentiation culture were subjected to heat stress of 37℃ and 43℃, respectively. For the 37℃ heat stress treatment, the culture conditions were the same as the differentiation culture except that the culture temperature was increased from 34℃ to 37℃. For the 43℃ heat stress treatment, SSCs were cultured in a 43℃ CO 2 incubator for 30 minutes every day and then returned to a 34℃ CO 2 incubator for further culture.

Total RNA-sequence and bioinformatics
We used RNA-Sequence to analyze gene expression for functional enrichment analysis [35], all DEGs were mapped to terms in the GO databases, and then signi cantly enriched GO terms were searched for among the DEGs using P < 0.05 as the threshold. GO term analysis was classi ed into three subgroups, namely biological process (BP), cellular component (CC) and molecular function (MF). All DEGs were mapped to the KEGG database, and searched for signi cantly enriched KEGG pathways at P < 0.05 level.

Statistical analysis
The dates are presented as the mean ± standard error of mean. The data were analyzed using one-way ANOVA. P≤0.05 was considered to indicate a statistically signi cant difference, and P≤0.01 was considered to indicate a highly signi cant difference among the different treatment groups.

Establishment of in vitro SSCs differentiation system
We successfully established the in vitro SSC differentiation culture system. We added SCF, BMP4, RA and Activing A to the SSC culture medium to induce SSCs differentiation and used real-time PCR to detect SSCs self-renewal and differentiation marker genes expression. The result showed compared with self-renewing SSCs cultured at 37℃, SSCs cultured for differentiation at 34℃ showed obvious colony-like growth on the 4th and 6th day after differentiation culture ( Fig. 1A). SSC marker genes ID4 and Thy-1 were signi cantly (P≤0.05) decreased at day 6 after differentiation culture. SSC differentiation marker gene c-kit, meiosis related genes Stra8 and Rec8, and spermatocyte related gene Sycp3 were signi cantly increased at day 6 after differentiation culture (Fig. 1B). These results showed that the SSCs differentiation system was successfully established.

Heat stress inhibited SSCs differentiation
In order to clarify the effect of high temperature on the SSC differentiation, the heat stress treatment on differentiated SSCs was carried out at 37℃and 43℃, respectively ( Fig. 2A). Firstly, we examined the effect of heat stress on SSCs marker genes expression during SSC differentiation. The results showed that 4 days after heat stress treatment, the expression of stem cell marker genes ID4 and Thy-1 in the 37℃and 43℃ heat shock-treated differentiation culture groups were signi cantly higher than those in the 34℃ differentiation culture group. Then, we examined the effects of heat stress on the expression of SSCs differentiation related genes. The results showed that 2 and 4 days after heat stress treatment, the expression of c-kit, Stra8, Rec8, Sycp3 and spermatocyte related gene Ovol1 in 37℃ and 43℃ differentiation groups were signi cantly lower than that in 34℃ differentiation group (P≤0.05). Finally, we compared the inhibitory effects of heat stress treatment at 37℃and 43℃ on SSC differentiation, and found that longterm heat stress treatment at 37℃ inhibited the differentiation of SSCs more signi cantly than heat stress treatment at 43℃ for 30min per day. Two and four days after heat stress treatment, the expression of differentiation related genes ckit, Stra8 and Rec8 in 37℃ treatment group was signi cantly lower than that in 43℃ treatment group, and the expression of stem cell marker genes Thy-1 was higher than that in 43℃ treatment group (Fig. 2B). These results showed that heat stress inhibited SSCs differentiation, and long-term 37℃ heat stress treatment inhibited SSC differentiation more signi cantly than 30 min 43℃ short-term heat stress treatment. In the subsequent experiments, we adopted long-term 37℃ heat stress to treat SSCs.

Heat stress changed gene expression of differentiation cultured SSCs
To reveal the molecular mechanism associated with the effect of heat stress treatment on SSCs differentiation, a DEG analysis was performed to identify gene expression changes between normal temperature (34℃) and heat stress temperature (37℃) during SSCs differentiation culture. The results showed that compared with SSCs cultured at 34℃, 765 genes were up-regulated and 895 genes were down-regulated in SSC cultured at 37℃ on day 2; On day 4, 3892 genes were up-regulated and 3360 genes down-regulated ( Fig. 3A and B). With the extension of heat stress treatment time from the second day to the fourth day, although the number of total expressed genes did not change signi cantly (29401 and 24713 respectively), the number of differentially expressed genes increased signi cantly (from 1160 to 7252) (Fig. 3C).
Gene ontology analysis of the differentially expressed genes Gene Ontology (GO) analysis was used to characterize the functions of the DEGs obtained from RNA-Seq. Three different aspects of DEGs called biological processes (BP), cellular component (CC) and molecular function (MF) re ected the effects of thermal stress on cell differentiation (Fig. 4A and B). The top 30 enriched terms on the second and fourth day after 37℃ heat stress treatment are shown in Fig. 4C and Fig. 4D. We compared the top 30 enriched terms on the second and fourth day after 37℃ heat stress treatment and found 11 common GO Terms (Fig. 4E). These 11 GO terms are cell adhesion molecule binding, rRNA binding, structural molecule activity, structural constituent of ribosome, large ribosomal subunit, cytosolic large ribosomal subunit, cytosolic part, ribosome, ribosomal subunit, cytosolic ribosome, and ribosome biogenesis ( Fig. 4C and D, Table 1).
KEGG analysis of the differentially expressed genes KEGG enrichment analysis of DEGs can reveal pathways with signi cant enrichment, which is helpful for nding signi cantly altered biological regulatory pathways. To further explore the roles of DEGs in the effect of SSCs differentiation after heat stress treatment, we tested whether the DEGs were enriched in certain KEGG pathways. The top 33 enriched KEGG pathways on the second and fourth after 37℃ heat stress treatment are shown in Fig. 5A and Fig. 5B. We compared the top 33 enriched KEGG pathway on the second and fourth day after 37℃ heat stress treatment and found 6 common KEGG pathways (Fig. 5C). These 6 common KEGG pathways are Ribosome, Carbon metabolism, Citrate cycle (TAC cycle), P53 signaling pathway, Bacterial invasion of epithelial cells and Apoptosis. In these 6 common KEGG pathway, only the Ribosome, Carbon metabolism and Citrate cycle (TAC cycle) were signi cantly enrich on the second day after 37℃ heat stress treatment ( Fig. 5A and B, Table 2).

Discussion
In this study, we successfully investigate the effect of heat stress on SSC differentiation by using in vitro differentiation cultured SSCs. The results show that high temperature inhibits SSC in vitro differentiation, and alter the expression of SSC transcriptome. RNA-seq analyses identify signi cantly inhibited pathways in DEGs after heat stress treatment, including p53 signaling pathway, carbon metabolism, and ribosome signaling pathways. These results provide new insights for the diagnosis and treatment of human oligospermia associated with high temperature.
Previous studies have shown that heat stress treatment induces the spermatogenic cell apoptosis in mice and rats, and leads to infertility gradually [36]. However, the effects of high temperature on SSCs development are still poorly understood due to the low number in testes. In this study, we added cytokines RA, BMP4, SCF and activin A into the SSC differentiation medium and established an in vitro SSC differentiation system [33]. Previous studies indicated that RA was su cient to induce the entry of SSCs into meiosis, and the expression of Stra8 was signi cantly increased 24h after induction of differentiation in combination with BMP4 [37,38]. As a ligand of c-kit, SCF binds to it and plays an important role in regulating the balance between self-renewal and differentiation of SSCs [39]. Activin A plays an crucial role in germ cell maturation during the period when gonocytes resume mitosis to form the SSCs and differentiating germ cell populations [40], and Activin A is widely used as a cytokine in stem cell differentiation cultured in vitro [41][42][43]. Through this culture system, we are able to study SSC differentiation process.
We evaluated the stem cell differentiation culture system by detecting the expression of spermatogenic stage-speci c marker genes [34]. The expression of the helix-loop-helix protein ID4 is selective for a subset of A single in mouse testes and plays a role in maintenance of the SSC pool [44]. The level of ID4 is predictive of stem cell or progenitor capacity in spermatogonia and dictates the interface of transition from the stem cell to the immediate progenitor state [45]. Flow cytometric cell sorting and the SSC transplantation assay demonstrated that Thy-1 is a unique surface marker of SSCs in neonatal pup, and adult testes of the mouse [46]. c-kit has been a marker for SSCs pluripotency lost. In early studies, c-kit expression is detected in type A (A 1 -A 4 ), intermediate, type B spermatogonia, as well as preleptotene spermatocytes, but not in the undifferentiated spermatogonia [47,48]. Stra8, as a response gene to RA, plays an important role in the initiation of meiosis during spermatogenesis and is a marker for germ cells to enter meiosis [49].
Rec8 is a key component of the meiotic cohesin complex. Rec8 has an essential role in mammalian meiosis, and both male and female Rec8 null mice have germ cell failure and sterility [50]. SYCP3 (or SCP3) is a DNA binding protein that forms a structural component of the ligand complex, which mediates chromosome binding or homologous pairing during meiosis in germ cells [51,52]. Ovol1 (previously known as movo1), encoding a member of the Ovo family of zinc-nger transcription factors, regulate meiotic pachytene progression during spermatogenesis by repressing Id2 expression, and the targeted deletion of Ovol1 leads to germ cell degeneration and defective sperm production in adult mice [53].
We successfully established SSC in vitro differentiation culture system. The results of this study showed that after we added RA, BMP4, SCF and activing A in SSC differentiation medium for differentiation culture, the expressions of ID4 and Thy-1 were down-regulated, while the expressions of c-kit, Stra8, Rec8, Sycp3 and Ovol1 were up-regulated, indicating that we successfully established the differentiation culture system of SSCs.
High temperature inhibited in vitro cultured SSC differentiation. In most male mammals, the temperature in the scrotum is usually 2-7 ℃ lower than the core body temperature, and the temperature of the testes is strictly regulated by a heat exchange system [54]. Therefore, we used 34℃ as the temperature for SSC in vitro differentiation culture. In previous studies, the 32-34.5℃was widely used for function SSC culture in vitro [55][56][57][58]. In this study, we used 37℃ or 43℃ as the heat stress temperature. 37℃ is the core body temperature, which is equivalent to the testicular temperature in patients with cryptorchidism. In many studies, 43℃ has been widely used as a heat stress treatment temperature to study the effects of high temperature on male germ cells in vivo [59]. In our previous study, 43℃was used as a heat stress temperature to treat self-renewal cultured SSCs in vitro, and it was found that heat stress treatment at 43℃ inhibited the differentiation of SSCs and did not induce SSC apoptosis [32]. The results of this study showed that both 37℃ and 43℃ heat stress inhibited SSC differentiation. The expression of SSC marker genes ID4 and Thy-1 increased signi cantly in differentiation cultured SSCs after heat stress treatment, and untill now no similar reports have been reported. After heat stress treatment, the expression of differentiation related genes c-kit, Stra8, Rec8, Sycp3 and Ovol1 in differentiation cultured SSCs was signi cantly decreased. Previous studies have shown that the expression of SYCP3 in testis of C57 adult mice was signi cantly decreased 1d and 7d after 43℃ 15min heat stress treatment [60, 61]. Stra8, which was periodically expressed in germ cells in seminiferous tubule, was signi cantly decreased after heat stress [62]. In our study, we also found that long-term heat stress treatment at 37℃ had a more obvious inhibitory effect on germ cell differentiation-related gene expression than the 30 min heat stress treatment at 43℃, which provided ideas for the pathogenesis of azoospermia caused by SSC differentiation disorder in cryptorchidism.
In this study, RNA-Seq analysis was performed on the in vitro differentiation cultured SSCs after heat shock treatment, and many DEGs were found. GO and KEGG analysis found that after heat stress treatment at 37℃, there were signi cant inhibition of some DEGs in p53 signal pathway, carbon metabolism and ribosome signal pathway. Previous studies suggest that p53 signaling pathway relate to cell differentiation closely [63, 64]. Jain AK's study show that in response to the differentiation stimuli such as RA, p53 is activated after being acetylated by CBP/p300 histone acetyl transferases to induce embryonic stem cells (ESCs) differentiation [65]. In our RNA-seq results, the Thrombospondins1 (Thbs1) gene in p53 signaling pathway was down-regulated. Thbs1 is a member of the extracellular matrix (ECM) protein family. Thbs1 is associated with angiogenic activity, endothelial cell migration and proliferation, and tumor angiogenesis Previous studies have shown that ribosome signaling pathway associated with cell differentiation. Sankaran's study found that ribosome levels selectively regulate translation and lineage commitment in human hematopoiesis [69]. The researchers note that a reduction in ribosome numbers leads to a reduction in the output of the GATA1 protein in blood stem cells, which in turn affects their differentiation into mature red blood cells [69]. The results of our study showed that 7 ribosome related GO terms were found in the 11 GO terms that were enriched on the second and fourth day of heat stress treatment at 37℃ in differentiation cultured SSCs. We found that Rpl13a, Rpl17, Rpl34, Rps28 and Rps2 genes were enriched in the 6 ribosomal related GO terms. KEGG results indicated that Rpl13a, Mrps18c, and Rps28 genes were enriched in ribosome signaling pathway. The results of our study indicate that ribosome signaling pathway may play an important role in inhibiting the differentiation of SSCs at high temperature.
The Carbon metabolism signaling pathways enriched in this study may play an important role in inhibiting SSCs differentiation at high temperature. For many years, stem cell metabolism was viewed as a byproduct of cell fate status rather than an active regulatory mechanism [70]. Carbon metabolism is a crucial aspect of cell life. Many studies have found that carbon metabolism is inseparable from cell differentiation. Both folate receptor 1 (folr1) overexpression and treatment with folinic acid stimulate β-cell differentiation in zebra sh and pig islets [71]. And the folic acid is an important vitamin of the one-carbon metabolism pathway that provides carbon units for numerous cellular processes [72,73]. Due to its essential role in nucleic acid synthesis, inhibition of folate metabolism blocks cellular proliferation [74]. Mitochondria are bioenergetic organelles that produce ATP via oxidative phosphorylation (OXPHOS) and play an important role in mediating stem cell fate and function. In the pre-implantation stage of mammalian development, cellular energy in the form of adenosine triphosphate (ATP) is generated primarily through the oxidation of carbon sources [75]. Loss of the mitochondrial complex III subunit rieske iron-sulfur protein (RISP) in fetal mouse hematopoietic stem cells allows them to proliferate but impairs their differentiation, leading to anemia and prenatal death [76]. Mitochondria dynamically regulate stem cell identity, self-renewal, and differentiation by orchestrating a transcriptional program [77].

Conclusion
These results indicate that high temperature at 37℃ signi cantly inhibits SSC differentiation and promotes enrichment of P53, ribosome and carbon metabolism signaling pathways in stem cell differentiation, providing a reference for the pathogenesis of heat-induced azoospermia.

Availability of data and materials
Please contact the corresponding authors for data requests.

Data availability statement
The datasets presented in this study can be found in online repositories.
Author contributions GWJ, LHX and FJ were responsible for the experiments, data analysis and editing of the manuscript. JH participated in the design of the study and edited the manuscript. MWZ was contributed to the conception, supervision and editing of the manuscript. All authors read and approved the nal manuscript.

Ethics approval
The experiments using mice were approved by the ethics committee of Ningxia Medical University, and all animal care and experiments were carried out in accordance with the institutional ethical guidelines for animal experiments.

Consent for publication
Not applicable  Figure 1 Establishment of in vitro SSC differentiation system. A. SSCs grew well in 37℃ self-renewal culture group and 34℃ differentiation culture group. Bar=100μm. B. SSC marker genes ID4 and Thy-1 were signi cantly decreased at day 4 and 6 after differentiation culture, and SSC differentiation marker gene c-kit and meiosis related genes Stra8, Rec8 and Sycp3 were signi cantly increased. Self-ren, self-renewal; 34℃diff, differentiation culture at 34℃; 34℃diff-4d, the fourth day of differentiation culture at 34℃; 34℃diff-6d, the sixth day of differentiation culture at 34℃. *P≤0.05, **P≤0.01.

Figure 2
Heat stress inhibited the SSC differentiation. A. In control group, SSC was cultured at 34℃. SSCs in heat stress treatment groups were cultured at 37℃ and 43℃, respectively. Bar=100μm. B. The expression of SSCs marker genes ID4 and Thy-1 in the 37℃ and 43℃ differentiation culture groups were signi cantly higher than those in the 34℃ differentiation culture group. The expression of SSCs differentiation marker gene c-kit, meiosis related genes Stra8 and Rec8, as well as spermatocyte leptotene and pachytene related genes Sycp3 and Ovol1 in 34℃ differentiation groups was signi cantly higher than those in 37℃ and 43℃differentiation groups.