Gonocyte Proliferation Research Articles

An in vitro three-dimensional (3D) testicular organoid culture system for efficient gonocyte maintenance and propagation using frozen/thawed neonatal bovine testicular tissues

Fertility preservation in prepubertal boys with cancer requires the cryopreservation of immature testicular tissues (ITTs) prior to gonadotoxic treatment. However, the limited number of germ cells in small human ITT biopsies necessitates the development of an in vitro culture system for germ cell expansion using frozen-thawed ITTs. Here, we generated testicular organoids for the in vitro maintenance and expansion of gonocytes from frozen-thawed two-week-old neonatal bovine ITTs. We investigated the effects of different cell-seeding densities, culture serums, seeding methods, and gonadotropin supplementations, on the maintenance and proliferation of enriched gonocytes. Our results demonstrated that enriched gonocytes and testicular cells from frozen-thawed neonatal ITTs could self-assemble into spheroid organoids in three days in an appropriate Matrigel-based culture environment. For the optimal formation of prepubertal testicular organoids, a seeding density of 1 × 106 cells/well is recommended over other densities. This strategy results in organoids with a mean diameter of 60.53 ± 12.12 μm; the mean number of organoids was 5.57 ± 1.60/105 μm2 on day 11. The viability of organoids was maintained at 79.75 ± 2.99% after being frozen and thawed. Supplementing the culture medium with glial cell-derived neurotrophic factor, fibroblast growth factor 2, and leukemia inhibitory factor, increased the proportion of KI67-positive proliferating cells in organoids, elevated the expression of C-KIT but reduced the expression of GFRα1 at day 28 when compared to those without hormone supplements (p< 0.05). In addition, supplementing the culture medium with follicle-stimulating hormone and testosterone helped to maintain a significantly higher viability (p< 0.05) in ITT organoids at day 28. These organoids could be cryopreserved for storage and thawed as needed. The successful generation of ITT organoids provides a valuable tool for establishing in vitro spermatogenesis, propagating human germ cells, investigating testicular physiology and the origin of germ cell tumors, and testing the toxicity of new drugs in future clinical applications.

Molecular characterization, expression patterns and cellular localization of BCAS2 gene in male Hezuo pig.

Breast carcinoma amplified sequence 2 (BCAS2) participates in pre-mRNA splicing and DNA damage response, which is implicated in spermatogenesis and meiosis initiation in mouse. Nevertheless, the physiological roles of BCAS2 in the testes of large mammals especially boars remain largely unknown. In this study, testes were collected from Hezuo pig at three development stages including 30 days old (30 d), 120 days old (120 d), and 240 days old (240 d). BCAS2 CDS region was firstly cloned using RT-PCR method, and its molecular characteristics were identified using relevant bioinformatics software. Additionally, the expression patterns and cellular localization of BCAS2 were analyzed by quantitative real-time PCR (qRT-PCR), Western blot, immunohistochemistry and immunofluorescence. The cloning and sequence analysis indicated that the Hezuo pig BCAS2 CDS fragment encompassed 678 bp open reading frame (ORF) capable of encoding 225 amino acid residues, and possessed high identities with some other mammals. The results of qRT-PCR and Western blot displayed that BCAS2 levels both mRNA and protein were age-dependent increased (p < 0.01). Additionally, immunohistochemistry and immunofluorescence results revealed that BCAS2 protein was mainly observed in nucleus of gonocytes at 30 d testes as well as nucleus of spermatogonia and Sertoli cells at 120 and 240 d testes. Accordingly, we conclude that BCAS2 is critical for testicular development and spermatogenesis of Hezuo pig, perhaps by regulating proliferation or differentiation of gonocytes, pre-mRNA splicing of spermatogonia and functional maintenance of Sertoli cells, but specific mechanism still requires be further investigated.

Cloning, expression analysis and localization of DAZL gene implicated in germ cell development of male Hezuo pig

Deleted in azoospermia-like (DAZL) is essential for mammalian testicular function and spermatogenesis. To explore the molecular characterization, expression patterns, and cellular localization of the DAZL in Hezuo pig testes, testicular tissue was isolated from Hezuo pig at five development stages including 30 days old (30 d), 90 days old (90 d), 120 days old (120 d), 180 days old (180 d), and 240 days old (240 d). DAZL cDNA was first cloned using the RT-PCR method, and its molecular characterization was analyzed using relevant bioinformatics software. Subsequently, the expression patterns and cellular localization of DAZL were evaluated using quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry. The cloning and sequence analysis showed that the Hezuo pig DAZL cDNA fragment contained 888 bp open reading frame (ORF) capable of encoding 295 amino acid residues and exhibited high identities with some other mammals. The qRT-PCR and Western blot results indicated that DAZL was specifically expressed in Hezuo pig testes, and DAZL levels of both mRNA and protein were expressed at all five reproductive stages of Hezuo pig testes, with extremely significant higher expression levels in 90 d, 120 d, 180 d, and 240 d than those in 30 d (p < 0.01). Additionally, immunohistochemistry results revealed that DAZL protein was mainly localized in gonocytes at 30 d testes, primary spermatocytes, and spermatozoon at other developmental stages, and Leydig cells throughout five development stages. Together, these results suggested that DAZL may play an important role by regulating the proliferation or differentiation of gonocytes, development of primary spermatocytes and spermatozoon, and functional maintenance of Leydig cells in testicular development and spermatogenesis of Hezuo pig. Nevertheless, the specific regulatory mechanisms underlying these phenomena still requires further investigated and verified.

Sperm-carried IGF2 downregulated the expression of mitogens produced by Sertoli cells: A paracrine mechanism for regulating spermatogenesis?

Insulin-like growth factor 2 (IGF2) mRNA has been found in human and mouse spermatozoa. It is currently unknown whether the IGF2 protein is expressed in human spermatozoa and, if so, its possible role in the cross-talk between germ and Sertoli cells (SCs) during spermatogenesis. To accomplish this, we analyzed sperm samples from four consecutive Caucasian men. Furthermore, to understand its role during the spermatogenetic process, porcine SCs were incubated with increasing concentrations (0.33, 3.33, and 10 ng/mL) of recombinant human IGF2 (rhIGF2) for 48 hours. Subsequently, the experiments were repeated by pre-incubating SCs with the non-competitive insulin-like growth factor 1 receptor (IGF1R) inhibitor NVP-AEW541. The following outcomes were evaluated: 1) Gene expression of the glial cell-line derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), and stem cell factor (SCF) mitogens; 2) gene and protein expression of follicle-stimulating hormone receptor (FSHR), anti-Müllerian hormone (AMH), and inhibin B; 3) SC proliferation. We found that the IGF2 protein was present in each of the sperm samples. IGF2 appeared as a cytoplasmic protein localized in the equatorial and post-acrosomal segment and with a varying degree of expression in each cell. In SCs, IGF2 significantly downregulated GDNF gene expression in a concentration-dependent manner. FGF2 and SCF were downregulated only by the highest concentration of IGF2. Similarly, IGF2 downregulated the FSHR gene and FSHR, AMH, and inhibin B protein expression. Finally, IGF2 significantly suppressed the SC proliferation rate. All these findings were reversed by pre-incubation with NVP-AEW541, suggesting an effect mediated by the interaction of IGF2 with the IGFR. In conclusion, sperm IGF2 seems to downregulate the expression of mitogens, which are known to be physiologically released by the SCs to promote gonocyte proliferation and spermatogonial fate adoption. These findings suggest the presence of paracrine regulatory mechanisms acting on the seminiferous epithelium during spermatogenesis, by which germ cells can influence the amount of mitogens released by the SCs, their sensitivity to FSH, and their rate of proliferation.

OR25-3 Sperm-carried IGF2 Downregulates Mitogens Released by Sertoli Cells: A Paracrine Mechanism of Spermatogenetic Regulation?

Abstract The presence of insulin-like growth factor 2 (IGF2) mRNA has been demonstrated in human and mouse spermatozoa. However, the expression of the IGF2 protein in spermatozoa and its function is not known. To accomplish this, we searched for IGF2 protein in spermatozoa of four healthy Caucasian men by Western blot (WB) and immunofluorescence (IM). Semen samples were collected by masturbation into a sterile container after 3-4 days of sexual abstinence. The spermatozoa were separated from leucocytes and epithelial cells by swim-up. The WB demonstrated the presence of the IGF2 protein in each sample examined. IM showed that IGF2 is a cytoplasmic protein with a variable degree of expression in each spermatozoon and localized in the equatorial and post-acrosomal segments. To evaluate the effects of IGF2, porcine Sertoli cells (SCs) were isolated from neonatal Large White pigs (7-15 days) using established methods. SCs were incubated with increasing concentrations (0.33, 3.33, and 10 ng/mL) of recombinant human IGF2 (rhIGF2) for 48 hours. At the end of incubation, the following endpoints were evaluated: a) expression of the GDNF, FGF2, and SCF genes, known to be mitogens that promote gonocyte proliferation and differentiation towards their spermatogonial fate, by real-time PCR (RT-PCR); b) gene (RT-PCR) and protein (WB and IM) expression of the follicle-stimulating hormone receptor (FSHR); and c) proliferation of SCs by flow cytometry. Subsequently, the effects of IGF2 were re-evaluated by pre-treating SCs with NVP-AEW541, a not competitive inhibitor of the insulin-like growth factor 1 receptor (IGF1R), known to be activated by IGF2. For this purpose, the NVP-AEW541 was added to the culture medium at a concentration of 1 µg/mL one hour before the IGF2 and was left for the entire duration of the incubation. The results of this study showed that IGF2 significantly downregulates GDNF gene expression in a concentration-dependent manner (-40.3% at 0.33 ng/mL, -48.2% at 3.33 ng/mL, -55.5%, respectively, p&amp;lt;0.01). Gene expression of FGF2 and SCF was significantly downregulated only by IGF2 at the highest concentration used (-23.6% and -39.1%, respectively, p&amp;lt;0.01). Similarly, IGF2 incubation downregulated FSHR mRNA (-27.1% at 0.33 ng/mL, -45.4.2% at 3.33 ng/mL, -21.1%, respectively, p&amp;lt;0.01) and protein (for both WB and IM) expression. Finally, SC proliferation significantly decreased after incubation with IGF2 (2.82±0.1% in untreated controls, -1.87±0.1% at 0.33 ng/mL, -2.19±0.2% at 3.33 ng/mL, and -2.36±0.1% at 10 ng/mL; p&amp;lt;0.01). In the presence of NVP-AEW541, all these effects were counteracted, thus suggesting that they are mediated by the IGF1R. In conclusion, these results suggest the presence of a paracrine regulatory mechanism between germ cells and SCs by which the former can influence the proliferation of SCs, their sensitivity to FSH, and their function by reducing the secretion of germ cell mitogens. Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 a.m.

Role of the Ubiquitin Ligase RNF149 in the Development of Rat Neonatal Gonocytes.

Male reproductive function depends on the formation of spermatogonial stem cells from their neonatal precursors, the gonocytes. Previously, we identified several UPS enzymes dynamically altered during gonocyte differentiation. The present work focuses on understanding the role of the RING finger protein 149 (RNF149), an E3 ligase that we found to be strongly expressed in gonocytes and downregulated in spermatogonia. The quantification of RNF149 mRNA from postnatal day (PND) 2 to 35 (puberty) in rat testis, brain, liver, kidney, and heart indicated that its highest levels are found in the testis. RNF149 knock-down in PND3 rat gonocytes was performed to better understand its role in gonocyte development. While a proliferative cocktail of PDGF-BB and 17β-estradiol (P+E) increased both the expression levels of the cell proliferation marker PCNA and RNF149 in mock cells, the effects of P+E on both genes were reduced in cells treated with RNF149 siRNA, suggesting that RNF149 expression is regulated during gonocyte proliferation and that there might be a functional link between RNF149 and PCNA. To examine RNF149 subcellular localization, EGFP-tagged RNF149 vectors were constructed, after determining the rat testis RNF149 mRNA sequence. Surprisingly, two variant transcripts were expressed in rat tissues, predicting truncated proteins, one containing the PA and the other the RING functional domains. Transfection in mouse F9 embryonal carcinoma cells and C18-4 spermatogonial cell lines showed differential subcellular profiles of the two truncated proteins. Overall, the results of this study support a role for RNF149 in gonocyte proliferation and suggest its transcription to variant mRNAs resulting in two proteins with different functional domains. Future studies will examine the respective roles of these variant proteins in the cell lines and isolated gonocytes.

Cullin4 E3 Ubiquitin Ligases Regulate Male Gonocyte Migration, Proliferation and Blood-Testis Barrier Homeostasis.

Ubiquitination, an essential posttranslational modification, plays fundamental roles during mammalian spermatogenesis. We previously reported the requirement of two Cullin 4 ubiquitin ligase family genes, Cullin 4a (Cul4a) and Cullin 4b (Cul4b), in murine spermatogenesis. Both genes are required for male fertility despite their distinct functions in different cell populations. Cul4a is required in primary spermatocytes to promote meiosis while Cul4b is required in secondary spermatocytes for spermiogenesis. As the two genes encode proteins that are highly homologous and have overlapping expression in embryonic germ cells, they may compensate for each other during germ cell development. In the present study, we directly address the potential functional redundancy of these two proteins by deleting both Cul4 genes, specifically, in the germ cell lineage during embryonic development, using the germ-cell specific Vasa-Cre line. Conditional double-knockout (dKO) males showed delayed homing and impaired proliferation of gonocytes, and a complete loss of germ cells before the end of the first wave of spermatogenesis. The dKO male germ cell phenotype is much more severe than those observed in either single KO mutant, demonstrating the functional redundancy between the two CUL4 proteins. The dKO mutant also exhibited atypical tight junction structures, suggesting the potential involvement of CUL4 proteins in spermatogonial stem cell (SSC) niche formation and blood–testis-barrier (BTB) maintenance. We also show that deleting Cul4b in both germ and Sertoli cells is sufficient to recapitulate part of this phenotype, causing spermatogenesis defects and drastically reduced number of mature sperms, accompanied by defective tight junctions in the mutant testes. These results indicate the involvement of CUL4B in maintaining BTB integrity.

RNA helicase DDX5 acts as a critical regulator for survival of neonatal mouse gonocytes.

ObjectivesMammalian spermatogenesis is a biological process of male gamete formation. Gonocytes are the only precursors of spermatogonial stem cells (SSCs) which develop into mature spermatozoa. DDX5 is one of DEAD‐box RNA helicases and expresses in male germ cells, suggesting that Ddx5 plays important functions during spermatogenesis. Here, we explore the functions of Ddx5 in regulating the specification of gonocytes.Materials and MethodsGerm cell‐specific Ddx5 knockout (Ddx5 ‐/‐) mice were generated. The morphology of testes and epididymides and fertility in both wild‐type and Ddx5 ‐/‐ mice were analysed. Single‐cell RNA sequencing (scRNA‐seq) was used to profile the transcriptome in testes from wild‐type and Ddx5 ‐/‐ mice at postnatal day (P) 2. Dysregulated genes were validated by single‐cell qRT‐PCR and immunofluorescent staining.ResultsIn male mice, Ddx5 was expressed in germ cells at different stages of development. Germ cell‐specific Ddx5 knockout adult male mice were sterile due to completely devoid of germ cells. Male germ cells gradually disappeared in Ddx5 ‐/‐ mice from E18.5 to P6. Single‐cell transcriptome analysis showed that genes involved in cell cycle and glial cell line‐derived neurotrophic factor (GDNF) pathway were significantly decreased in Ddx5‐deficient gonocytes. Notably, Ddx5 ablation impeded the proliferation of gonocytes.ConclusionsOur study reveals the critical roles of Ddx5 in fate determination of gonocytes, offering a novel insight into the pathogenesis of male sterility.

HnRNPU in Sertoli cells cooperates with WT1 and is essential for testicular development by modulating transcriptional factors Sox8/9.

Background: Sertoli cells are essential regulators of testicular fate in the differentiating gonad; however, its role and underlying molecular mechanism of regulating testicular development in prepubertal testes are poorly understood. Although several critical regulatory factors of Sertoli cell development and function have been identified, identifying extrinsic factors that regulate gonocyte proliferation and migration processes during neonatal testis development remains largely unknown.Methods: We used the Sertoli cell-specific conditional knockout strategy (Cre/Loxp) in mice and molecular biological analyses (Luciferase assay, ChIP-qPCR, RNA-Seq, etc.) in vitro and in vivo to study the physiological roles of hnRNPU in Sertoli cells on regulating testicular development in prepubertal testes.Results: We identified a co-transcription factor, hnRNPU, which is highly expressed in mouse and human Sertoli cells and required for neonatal Sertoli cell and pre-pubertal testicular development. Conditional knockout of hnRNPU in murine Sertoli cells leads to severe testicular atrophy and male sterility, characterized by rapid depletion of both Sertoli cells and germ cells and failure of spermatogonia proliferation and migration during pre-pubertal testicular development. At molecular levels, we found that hnRNPU interacts with two Sertoli cell markers WT1 and SOX9, and enhances the expression of two transcriptional factors, Sox8 and Sox9, in Sertoli cells by directly binding to their promoter regions. Further RNA-Seq and bioinformatics analyses revealed the transcriptome-wide of key genes essential for Sertoli cell and germ cell fate control, such as biological adhesion, proliferation and migration, were deregulated in Sertoli cell-specific hnRNPU mutant testes.Conclusion: Our findings demonstrate an essential role of hnRNPU in Sertoli cells for prepubertal testicular development and testis microenvironment maintenance and define a new insight for our understanding of male infertility therapy.

Cyclin A2 is essential for mouse gonocyte maturation

ABSTRACT In mammals, male gonocytes are derived from primordial germ cells during embryogenesis, enter a period of mitotic proliferation, and then become quiescent until birth. After birth, the gonocytes proliferate and migrate from the center of testicular cord toward the basement membrane to form the pool of spermatogonial stem cells (SSCs) and establish the SSC niche architecture. However, the molecular mechanisms underlying gonocyte proliferation, migration and differentiation are largely unknown. Cyclin A2 is a key component of the cell cycle and required for cell proliferation. Here, we show that cyclin A2 is required in mouse male gonocyte development and the establishment of spermatogenesis in the neonatal testis. Loss of cyclin A2 function in embryonic gonocytes by targeted gene disruption affected the regulation of the male gonocytes to SSC transition, resulting in the disruption of SSC pool formation, imbalance between SSC self-renewal and differentiation, and severely abnormal spermatogenesis in the adult testis.

Cloning, Molecular Characterization and Expression Patterns of DMRTC2 Implicated in Germ Cell Development of Male Tibetan Sheep.

The double sex and mab-3-related transcription factors like family C2 (DMRTC2) gene is indispensable for mammalian testicular function and spermatogenesis. Despite its importance, what expression and roles of DMRTC2 possesses and how it regulates the testicular development and spermatogenesis in sheep, especially in Tibetan sheep, remains largely unknown. In this study, DMRTC2 cDNA from testes of Tibetan sheep was firstly cloned by the RT-PCR method, and its molecular characterization was identified. Subsequently, the expression and localization patterns of DMRTC2 were evaluated by quantitative real-time PCR (qPCR), Western blot, and immunofluorescence. The cloning and sequence analysis showed that the Tibetan sheep DMRTC2 cDNA fragment contained 1113 bp open reading frame (ORF) capable of encoding 370 amino acids, and displayed high identities with some other mammals, which shared an identical DM domain sequence of 47 amino acids ranged from residues 38 to 84. qPCR and Western blot results showed that DMRTC2 was expressed in testes throughout the development stages while not in epididymides (caput, corpus, and cauda), with higher mRNA and protein abundance in Tibetan sheep testes of one- and three-year-old (post-puberty) compared with that of three-month-old (pre-puberty). Immunofluorescence results revealed that immune staining for DMRTC2 protein was observed in spermatids and spermatogonia from post-puberty Tibetan sheep testes, and gonocytes from pre-puberty Tibetan sheep testes. Together, these results demonstrated, for the first time, in sheep, that DMRTC2, as a highly conserved gene in mammals, is essential for sheep spermatogenesis by regulating the proliferation or differentiation of gonocytes and development of spermatids in ram testes at different stages of maturity.

Gonad formation and pre-meiotic proliferation of gonocytes in the Japanese willow minnow Gnathopogon caerulescens

Gonad formation and pre-meiotic proliferation of gonocytes in the Japanese willow minnow Gnathopogon caerulescens

Cyclooxygenase 2 (COX2) expression and prostaglandin synthesis in neonatal rat testicular germ cells: Effects of acetaminophen and ibuprofen.

In infants, fever is often treated with acetaminophen or ibuprofen, two antipyretic and analgesic drugs inhibiting cyclooxygenases (COXs), enzymes catalyzing prostaglandin production. Infancy represents a critical developmental period when neonatal germ cells/gonocytes differentiate to spermatogonial stem cells required for spermatogenesis. (a) Determine the expression of Cox2 and associated genes in postnatal day (PND)3 rat gonocytes compared to spermatogonia. (b) Examine whether acetaminophen or ibuprofen disrupts neonatal gonocyte functions. (c) Determine whether neonatal gonocytes produce prostaglandins and whether this process is altered by acetaminophen and ibuprofen. The expression of Cox2 and related genes was determined by gene arrays and qPCR. Cox2 protein levels were determined by immunocyto/histochemistry and immunoblots. The effects of acetaminophen and ibuprofen on PND3 gonocyte viability, apoptosis, proliferation, and differentiation were examined alone and with a proliferation cocktail or differentiation factor. Prostaglandins were examined by immunocyto/histochemistry and LC-MS. Cox2 and related genes are highly expressed in gonocytes and spermatogonia. Acetaminophen and ibuprofen did not affect gonocyte survival or apoptosis, but they increased gonocyte proliferation. Ibuprofen significantly reduced RA-induced Stra8 expression, indicating an inhibitory effect on differentiation. Ibuprofen combined with RA decreased Cox2 mRNA and protein expression. PGE2 and PGF2α were produced by neonatal gonocytes and decreased by acetaminophen and ibuprofen. The concomitant decrease of Stra8 expression, Cox2 expression, and PGE2 and PGF2a production in gonocytes co-treated with RA suggests that Cox2 plays a role in PND3 gonocyte differentiation. The effects of acetaminophen and ibuprofen on proliferation suggest a negative relationship between Cox2 and proliferation. Treating neonates with acetaminophen or ibuprofen could disrupt gonocyte development, leading to adverse reproductive effects. Understanding COX2 role in neonatal gonocytes and the potential risk of acetaminophen and ibuprofen treatment of infants may help prevent male reproductive pathologies.

Expression and cellular localization of double sex and mab-3 related transcription factor 1 in testes of postnatal Small-Tail Han sheep at different developmental stages

Double sex and mab-3 related transcription factor 1 (Dmrt1), an evolutionarily conserved gene, is a sex-related gene expressed in male gonads, that is involved in the regulation of sex differentiation, testicular development and reproductive function maintenance. Until now, functional studies on the Dmrt1 gene in sheep (Ovis aries) have been lacking. In this study, testis, heart, liver, spleen, lung, kidney and longissimus dorsi muscle tissues were collected from Small-Tail Han sheep at 0, 2, 5, 12 and 24months after birth (mab). Dmrt1 expression and cellular localization were detected in various testicular tissues by quantitative real time PCR (qRT-PCR), western blot and immunohistochemistry methods. The morphological structures of testicular tissues at different developmental stages were observed by hematoxylin & eosin (HE) staining. The Dmrt1 mRNA expression levels in 12 and 24 mab sheep were significantly higher than those in 0 and 2 mab sheep (P<0.05), and Dmrt1 protein expression showed a similar trend. The qRT-PCR results in various tissues at 12 mab showed that Dmrt1 mRNA was predominantly expressed in testes. Immunohistochemical staining in testes at different developmental stages showed that Dmrt1 protein immunoreactive responses were mainly localized in Sertoli cells and gonocytes at 0, 2 and 5 mab, while they were localized in spermatocytes, sperm cells and some spermatogonia and Sertoli cells at 12 and 24 mab. We speculate that the Dmrt1 gene plays a vital role in postnatal sheep spermatogenesis, perhaps by regulating the maturation and functional maintenance of Sertoli cells, the proliferation and differentiation of gonocytes in prepubertal sheep testes, and the mitosis and meiosis of germ cells in adult sheep, but the specific mechanisms underlying these phenomena must be further studied and verified. AbbreviationsImage 1BCAbicinchoninic acidcDNADNA complementary to RNADABdiaminobenzidineDmrt1double sex and mab-3 related transcription factor 1FOXL2forkhead transcription factor gene 2HEhematoxylin-eosinHRPhorseradish peroxidasePBSphosphate buffer salinePlzfpromyelocytic leukemia zinc fingerPVDFpolyvinylidene fluorideqRT-PCRquantitative real time PCRRAretinoic acidRspo1R-spondin1SDS-PAGEsodium dodecyl sulfate polyacrylamide gel electrophoresisSohlh1spermatogenesis and oogenesis-specific basic helix-loop-helix1SOX9sex determining region Y-box 9Srysex-determining region of the Y chromosomeSSCsspermatogonial stem cellsStra8stimulated by retinoic acid 8

Requirement for CCNB1 in mouse spermatogenesis

Spermatogenesis, which involves mitosis and meiosis of male germ cells, is a highly complicated and coordinately ordered process. Cyclin B1 (CCNB1), an important regulator in cell cycle machinery, is proved essential for mouse embryonic development. However, the role of CCNB1 in mammalian spermatogenesis remains unclear. Here we tested the requirement for CCNB1 using conditional knockout mice lacking CCNB1 in male germ cells. We found that ablation of CCNB1 in gonocytes and spermatogonia led to mouse sterile caused by the male germ cells’ depletion. Gonocyte and spermatogonia without CCNB1 is unable to proliferate normally and apoptosis increased. Moreover, CCNB1 ablation in spermatogonia may promote their differentiation by downregulating Lin28a and upregulating let-7 miRNA. However, ablation of CCNB1 in premeiotic male germ cells did not have an effect on meiosis of spermatocytes and male fertility, suggesting that CCNB1 may be dispensable for meiosis of spermatocytes. Collectively, these results indicate that CCNB1 is critically required for the proliferation of gonocytes and spermatogonia but may be redundant in meiosis of spermatocytes in mouse spermatogenesis.

Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells.

Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology.

Maternal obesity disturbs the postnatal development of gonocytes in the rat without impairment of testis structure at prepubertal age

In this study, we evaluated whether maternal obesity (MO) affects testis development and gonocyte differentiation in the rat from 0.5 to 14.5 postnatal days. Male Wistar rats were used at 0.5, 4.5, 7.5, and 14.5 days post partum (dpp). These rats were born from obese mothers, previously fed with a high-fat diet (20% saturated fat), for 15 weeks, or normal mothers that had received a balanced murine diet (4% lipids). MO did not affect testis weight or histology at birth but changed the migratory behavior of gonocytes. The density of relocated cells was higher in MO pups at 0.5 dpp, decreased at 4.5 dpp, and differed from those of control pups, where density increased exponentially from 0.5 to 7.5 dpp. The numerical density of gonocytes within seminiferous cords did not vary in MO, in relation to control neonates, for any age considered, but the testis weight was 50% lower at 4.5 dpp. A wide variation in plasmatic testosterone and estrogen levels was observed among the groups during the first week of age and MO pups exhibited higher steroid concentrations at 4.5 dpp, in comparison with controls. At this age, higher estrogen levels of MO pups impaired the gonocyte proliferation. At 7.5 dpp, the testicular size and other parameters of gonocyte development are retrieved. In conclusion, MO and saturated lipid diets disturb gonocyte development and sexual steroid levels during the first days of life, with recovery at prepubertal age.

The chemokine CXCL12 and its receptor CXCR4 are implicated in human seminoma metastasis

Seminoma and non-seminoma tumours increasingly occur within the western population. These tumours originate from carcinoma in situ (CIS) cells, which arise from dysfunctional gonocytes. CXCL12 and its receptors, CXCR4 and CXCR7, have been implicated in migration, proliferation and survival of gonocytes and their precursors and progeny, primordial germ cells and spermatogonial stem cells respectively. We previously found evidence that several miRNA molecules predicted to modulate CXCR4 signalling are differentially expressed during the differentiation of gonocytes into spermatogonia in mice. Bioinformatic analysis predicted these miRNA to modulate CXCR4 signalling, leading us to hypothesize that CXCL12-mediated CXCR4 signalling is involved in the disrupted differentiation of gonocytes that underpins CIS formation. Indeed, we detected CXCL12 in Sertoli cells of normal human testis, and relatively high expression in tumour stroma with concomitant weak staining in dispersed tumour cells. In contrast, CXCR4 was expressed in spermatogonial and meiotic germ cells of normal testis and in the majority of tumour cells. Quantitative RT-PCR identified elevated CXCR4 transcript levels in seminoma compared with normal testis and to non-seminoma, potentially reflecting the higher proportion of dysfunctional germ cells within seminomas. In the normal testis, expression of CXCR4 downstream signalling molecules phospho-MEK1/2 and phospho-ERK1/2 correlated with CXCR4/CXCL12 expression. Strikingly, this correlation was absent in seminoma and non-seminoma samples, suggesting that CXCL12 signalling is disrupted. Proliferation rate and cell survival were not altered by CXCL12 in either seminoma (TCam-2) or non-seminoma (833ke) cell lines. However, CXCL12 exposure induced TCam-2 cell invasion though simulated basement membrane, while in contrast, we provide the novel evidence that CXCR4-expressing non-seminoma cell lines 833ke and NTera2/D1 do not invade in response to CXCL12. These findings indicate that CXCL12 expression in the human testis may selectively influence seminoma migration and metastasis, correlating with its importance in gonocyte and spermatogonial stem cell biology.

Effects of extracellular matrices and lectin Dolichos biflorus agglutinin on cell adhesion and self-renewal of bovine gonocytes cultured in vitro

Surface molecules of primitive male germ cells, gonocytes, are essential components for regulating cell adhesion and maintaining self-renewal in mammalian species. In domestic animals, the stage-specific glycan epitope α-N-acetylgalactosamine (GalNAc) is recognised by the lectin Dolichos biflorus agglutinin (DBA) and is found on the surface of gonocytes and spermatogonia. Gonocytes from bovine testis formed mouse embryonic stem-like cell colonies on plates that had been coated with DBA or extracellular matrix (ECM) components, such as gelatin (GN), laminin (LN) and poly-L-lysine (PLL). The number of colonies on the DBA-coated plate was significantly higher than that on the GN-, LN- and PLL-coated plates. Pretreating gonocytes with DBA to neutralise the terminal GalNAc residues strongly suppressed colony formation. Furthermore, expression of a germ cell-specific gene and pluripotency-related transcription factors was increased considerably on the DBA-coated plates. These results suggest that the GalNAc residues on gonocytes can recognise precoated DBA on plates and the resulting GalNAc-DBA complexes support germ cell and stem cell potentials of gonocytes in vitro. These glycan complexes, through the GalNAc epitope, may provide a suitable microenvironment for the adhesion and cell proliferation of gonocytes in culture.

Gonocyte development in rats: proliferation, distribution and death revisited

Spermatogonial stem cells are responsible for the constant production of spermatozoa. These cells differentiate from the gonocytes, but little is known about these cells and their differentiation into spermatogonia. This study analyzed rat gonocyte proliferation, death and distribution as well as their differentiation into spermatogonia. Rat testes were collected at 19 dpc and at 1, 3, 5, 8, 11 and 15 dpp and submitted to apoptosis investigation through morphological analysis and TUNEL, p53 and cleaved caspase 3 labeling. Ki67 and MVH labeling was used to check gonocyte proliferation and quantification, respectively. OCT4 and DBA labeling were used to check gonocyte differentiation. Seminiferous cord length and gonocyte numerical density were measured to check gonocyte distribution along the seminiferous cords. Although a reduction of gonocyte number per testicular section has been observed from 1 to 5 dpp, the total number of these cells did not change. Apoptotic gonocytes were not detected at these ages, suggesting that the reduction in gonocyte number per testicular section was due to their redistribution along the seminiferous cords, which showed continuous growth from 19 dpc to 5 dpp. The first proliferating germ cells were observed at 8 dpp, coinciding with OCT4 upregulation and with the emergence of the first spermatogonia. In conclusion, this study suggests that (a) gonocytes do not die in the first week after birth, but are rather redistributed along the seminiferous cords just before their differentiation into spermatogonia; (b) mitosis resumption and the emergence of the first spermatogonia are coincident with OCT4 upregulation.