ERK/CREB and p38 MAPK/MMP14 Signaling Pathway Influences Spermatogenesis through Regulating the Expression of Junctional Proteins in Eriocheir sinensis Testis.

The hemolymph-testis barrier (HTB) is a reproduction barrier in Crustacea, guaranteeing the safe and smooth process of spermatogenesis, which is similar to the blood-testis barrier (BTB) in mammals. The MAPK signaling pathway plays an essential role in spermatogenesis and maintenance of the BTB. However, only a few studies have focused on the influence of MAPK on crustacean reproduction. In the present study, we knocked down and inhibited MAPK in Eriocheir sinensis. Increased defects in spermatogenesis were observed, concurrently with a damaged HTB. Further research revealed that es-MMP14 functions downstream of ERK and p38 MAPK and degrades junctional proteins (Pinin and ZO-1); es-CREB functions in the ERK cascade as a transcription factor of ZO-1. In addition, when es-MMP14 and es-CREB were deleted, the defects in HTB and spermatogenesis aligned with abnormalities in the MAPK. However, JNK impacts the integrity of the HTB by changing the distribution of intercellular junctions. In summary, the MAPK signaling pathway maintains HTB integrity and spermatogenesis through es-MMP14 and es-CREB, which provides insights into the evolution of gene function during barrier evolution.

Regulation of spermatogonial stem cell self-renewal and proliferation in mammals.

The generation of functional sperm relies on spermatogonial stem cells (SSCs) as they can maintain a stem cell pool for continuous generation of functional spermatozoa. The maintenance of SSCs is regulated by several factors. In this paper, we summarize the niche and intrinsic factors in regulating SSC self-renewal and proliferation. GDNF regulates SSC self-renewal through Ras-ERK1/2, SFC, PI3K/Akt and MEK/ERK-mTOR signaling pathways. FGF activates MAPK2K1, ERK and Akt pathways and EGF activates ERK and Akt pathways to induce SSC proliferation. Wnt ligands regulate SSC self-renewal and proliferation through both ß-catenin dependent and independent pathways. SCF1 and CXCL12 are also found to have roles in SSC maintenance. As for intrinsic factors in SSCs, ETV5, Bcl6b, Lhx1, ID4 and Nanos2 are regulated by niche factors. They act as the downstream factors of niche factors in regulating SSC self-renewal and proliferation. Transcriptional factors OCT4 and PLZF, as well as FOXO1 in SSCs can directly regulate SSC self-renewal and proliferation. Although we have identified the factors, the detailed mechanism of these factors in regulating SSC fate determination is largely unknown. Here, we summarize factors which have roles in SSC fate determination and hope it will be beneficial for further study and treatment of male infertility.

The PI3K/AKT signaling pathway: How does it regulate development of Sertoli cells and spermatogenic cells?

The PI3K/AKT signaling pathway is one of the most crucial regulatory mechanisms in animal cells, which can mainly regulate proliferation, survival and anti-apoptosis in cell lines. In the seminiferous epithelium, most studies were concentrated on the role of PI3K/AKT signaling in immature Sertoli cells (SCs) and spermatogonia stem cells (SSCs). PI3K/AKT signaling can facilitate the proliferation and anti-apoptosis of immature Sertoli cells and spermatogenic cells. Besides, in mature Sertoli cells, this pathway can disintegrate the structure of the blood-testis barrier (BTB) via regulatory protein synthesis and the cytoskeleton of Sertoli cells. All of these effects can directly and indirectly maintain and promote spermatogenesis in male testis.

PIWIs maintain testis apoptosis to remove abnormal germ cells in Eriocheir sinensis.

PIWI proteins play important roles in germline development in the mammals. However, the functions of PIWIs in crustaceans remain unknown. In the present study, we identified three Piwis from the testis of Eriocheir sinensis (E. sinensis). Three Piwi genes encoded proteins with typical features of PIWI subfamilies and were highly expressed in the testis. Three PIWIs could be detected in the cytoplasm of spermatocytes and spermatids, while in spermatozoa, we could only detect PIWI1 and PIWI3 in the nucleus. The knockdown of PIWIs by dsRNA significantly affected the formation of the nuclei in spermatozoa, which resulted in deviant and irregular nuclei. PIWI defects significantly inhibited the apoptosis of abnormal germ cells through the caspase-dependent apoptosis pathway and p53 pathway. Knockdown of PIWIs inhibited the expression of caspase (Casp) 3, 7, 8, and p53 without affecting Bcl2 (B-cell lymphoma gene 2), Bax (B-cell lymphoma-2-associated X), and BaxI (B-cell lymphoma-2-associated X inhibitor), which further significantly increased abnormal spermatozoa in the knockdown-group crabs. These results show a new role of PIWI proteins in crustaceans that is different from that in mammals. In summary, PIWIs play roles in the formation of the germline nucleus and can maintain apoptosis in abnormal germ cells to remove abnormal germ cells in E. sinensis.

Conversion from spermatogonia to spermatocytes: Extracellular cues and downstream transcription network.

Spermatocyte (spc) formation from spermatogonia (spg) differentiation is the first step of spermatogenesis which produces prodigious spermatozoa for a lifetime. After decades of studies, several factors involved in the functioning of a mouse were discovered both inside and outside spg. Considering the peculiar expression and working pattern of each factor, this review divides the whole conversion of spg to spc into four consecutive development processes with a focus on extracellular cues and downstream transcription network in each one. Potential coordination among Dmrt1, Sohlh1/2 and BMP families mediates Ngn3 upregulation, which marks progenitor spg, with other changes. After that, retinoic acid (RA), as a master regulator, promotes A1 spg formation with its helpers and Sall4. A1-to-B spg transition is under the control of Kitl and impulsive RA signaling together with early and late transcription factors Stra8 and Dmrt6. Finally, RA and its responsive effectors conduct the entry into meiosis. The systematic transcription network from outside to inside still needs research to supplement or settle the controversials in each process. As a step further ahead, this review provides possible drug targets for infertility therapy by cross-linking humans and mouse model.

Bone morphogenetic protein 2 (BMP2) mediates spermatogenesis in Chinese mitten crab Eriocheir sinensis by regulating kinesin motor KIFC1 expression.

The TGF-beta superfamily is widely involved in cell events such as cell division and differentiation, while bone morphogenetic proteins (BMPs) belong to one of the subgroups. Their functions in crustacean spermatogenesis are still unknown. In this study, we first identified the bone morphogenetic protein 2 (bmp2) from Eriocheir sinensis (E. sinensis) testis. The es-BMP2 shows high expression in E. sinensis testis. We found that es-BMP2 is expressed in spermatids. The successfully knockdown of es-BMP2 through in vivo RNAi are used for functional analysis. Compared with the control group, the proportion of abnormal nuclear cup morphology in mature spermatozoa increased significantly after es-bmp2 RNAi, suggesting that es-BMP2 plays an important role in mature sperm morphogenesis. Immunofluorescence results confirm this finding. In order to study the specific mechanism of es-BMP2 involved in spermiogenesis, we tested kinesin-14 KIFC1, which functions in the nucleus formation of spermatozoa in E. sinensis. The results showed that knockdown of es-BMP2 caused a significant decrease of es-KIFC1 expression. We further performed es-bmp2 knockdown in vitro in primary cultured testis cells. es-KIFC1 expression was significantly reduced after es-bmp2 RNAi. The above results indicate that es-BMP2 participates in maintaining the spermiogenesis of E. sinensis by regulating es-KIFC1 expression.