G-protein-coupled receptor 39 (GPR39) plays a role in cellular and physiological processes, including insulin secretion, cell death inhibition, wound healing, and obesity. Increasing evidence suggests that GPR39 is potently stimulated by zinc ions (Zn2+) and is therefore considered a putative Zn2+ receptor. Given the importance of Zn2+ in the reproductive system, we proposed that GPR39 might have a functional role in the reproductive system. However, the localization of GPR39 in the reproductive system remains unknown. Here, we used mice expressing a Gpr39 promoter-driven LacZ reporter system to detect Gpr39 expression in the reproductive system at different phases of the estrous cycle and found an interesting region-specific distribution of Gpr39 in the mouse oviduct epithelium, with strong expression at the ampulla and weak expression at the isthmus, which was consistent with the results using reverse transcription polymerase chain reaction and immunofluorescence. Moreover, using ZnSeAMG staining, we found that Zn2+, the putative ligand of GPR39, also found a distribution similar to GPR39 expression, suggesting that their potential interaction mediates fertilization and embryo transportation.

Once considered a simple medium for sperm and embryo transport, the functional spectrum of uterine fluid is now expanding. Novel molecular players, such as extracellular vesicles and mobile RNAs, have been detected in the uterine fluid of livestock, rodents, and humans. These novel molecules, together with previously known ions and proteins, ensure uterine fluid homeostasis and facilitate embryo–maternal interactions. Here, we propose that these molecules may also carry information that mirrors maternal environmental exposure and possibly relay such information to the embryo via uterine fluid, generating long-term epigenetic effects on the offspring via embryonic and placental programming. Moreover, the development of systematic profiling of uterine fluid molecular signatures may now hold promise, relying on high-throughput methods and non-invasive biomarkers for clinical use.

In mammalian species, the window of implantation is defined as a limited period when implantation-competent blastocyst can interact with the receptive endometrium1. Besides the acquisition of blastocyst competency and uterine receptivity, initiation of embryo implantation also involves a timely reabsorption of intrauterine fluids, which facilitates the apposition of embryo with the uterine lining2. These processes are coordinated by preimplantation ovarian estrogen (E2) and progesterone (P4): a small E2 surge imposed on a P4-primed environment initiates implantation1, where the level of E2 and ratio of P4/E2 must be kept within an optimal range3,4. To date, numerous genes regulating uterine receptivity and blastocyst implantation have been identified using transgenic mouse models5. However, genes controlling uterine fluid homeostasis are much less explored, and their pathophysiological significance in embryo implantation remains poorly understood. In the present study, combining genetic and pharmacological approaches, we demonstrate that an excess of intrauterine fluids caused by increased expression of two water channel genes, aquaporin-5 (Aqp5) and -8 (Aqp8), is a major contributor to abnormal implantation in pregnant mice treated with supraphysiological doses of E2. We also showed that P4 administration neutralized E2-induced Aqp5/8 overexpression, preventing excessive intrauterine fluid accumulation and improving implantation.During the in vitro fertilization/embryo transfer (IVF/ET) procedure, the standard ovarian hyperstimulation could cause supraphysiological levels of steroid hormones such as E2, leading to altered E2/P4 ratio and subsequently impaired implantation4. As uterine fluid homeostasis is dynamically regulated by sterioid hormones (E2 and P4)2 and an excess of intrauterine fluids at preimplantation causes abnormal implantation6,7, we hypothesized that abnormal uterine fluid secretion caused by elevated E2 levels may contribute to the defective implantation and aimed to identify the underlying mechanisms. To investigate the influence of supraphysiological E2 levels on embryo implantation, we treated pregnant mice with E2 (with different dosages, Figure 1A) on day 4 of pregnancy (08:30), and found that a single injection of > 50 ng of E2 efficiently disrupted embryo implantation as examined on day 5 and day 6, showing both delayed implantation and aberrant embryo spacing (Figure 1A-1C). The aberrant implantation indeed led to increased pregnancy loss at midgestation and decreased litter size on day 12 of pregnancy (Figure 1D and Supplementary information, Figure S1A-S1D), consistent with previous reports that aberrant timing and site of implantation caused adverse ripple effects for the ongoing pregnancy8,9,10. We next used this hyper-estrogen-induced pathophysiological mouse model to explore the role of excessive uterine fluid accumulation in the defective implantation. To estimate the volume of intrauterine fluids in mice, the cervix end of a uterine horn was first ligated (on day 3 08:30) to prevent fluid leakage. Twenty-four hours later the mice were treated with either E2 (100 ng) or vehicle (oil) for 8 h, and then the uteri from both groups were dissected for luminal fluid volume measurement (Figure 1E and Supplementary information, Figure S1E). The volume of intrauterine fluids was estimated by the size of the fluid-drop stain on a filter paper (Figure 1F and Supplementary information, Data S1). The uteri from E2-treated mice showed a dramatic increase in the intrauterine fluid volume, while the percentage of water weight within the uterine tissue of E2-treated mice (after releasing the luminal fluid) was similar to that of the control group (Figure 1G), suggesting that fluid retention occurred in the uterine lumen but not within the stroma, which is most likely due to increased fluid net flow into the lumen upon changed interstitial versus intraluminal osmolarity. We further examined the implantation status after performing the same ligation procedure (on day 3) followed by E2 or vehicle treatment (on day 4) (Supplementary information, Figure S1F), and found that although the ligation procedure did not hamper embryo implantation in vehicle-treated control group, it enhanced the adverse effect of E2 treatment and caused almost complete implantation failure in the ligated uterine horn (Figure 1H and 1I). These results clearly demonstrated that hyper-estrogen-induced excessive intrauterine fluid retention is an important contributor to defective implantation.To explore the underlying mechanism for the substantial increase in luminal fluid volume, we performed microarray analyses of E2- and vehicle-treated uteri and searched for potential genes responsible for the drastic intrauterine fluid increase. Among the possible candidates, two water channel genes, Aqp5 and Aqp 8, showed a simultaneous increase in expression levels. As aquaporin-mediated fluid transport is involved in a wide range of physiological and pathological conditions11, and previous studies have shown that the expression of aquaporins is dynamically regulated in the uterus during pregnancy12,13,14, we next examined the uterine expression levels of aquaporin family members in day-4 uteri (untreated, treated with E2 or ER antagonist ICI 182780) by RT-PCR analysis and confirmed the drastic increase in the expression levels of Aqp5 and Aqp8 after E2 treatment (Figure 1J). A weak expression of Aqp5 was observed in the absence of E2 treatment, which was inhibited by ICI treatment (Figure 1J). Similar results were obtained using real-time PCR analysis (Figure 1K and 1L). The expression levels of Aqp5/8 showed a gradual increase, which coincides with the increase in the volume of intrauterine fluids after E2 treatment (Supplementary information, Figure S1G-S1I). We next performed immunofluorescence analysis for Aqp5 and Aqp8 proteins (antibody specificity was confirmed in positive tissues, Supplementary information, Figure S1J and S1K) in day-4 uteri and observed an enhanced staining of Aqp5 and Aqp8 after E2 treatment (Figure 1M). Notably, both Aqp5 and Aqp8 proteins are specifically localized on the apical surface, the secretion side of the uterine glandular epithelium (Figure 1M), suggesting an active role in E2-induced luminal fluid secretion.To examine whether Aqp5 and Aqp8 are responsible for the E2-induced excessive luminal fluid accumulation, we bred Aqp5−/−, Aqp8−/− and Aqp5−/−Aqp8−/− mice, and mated the female knockout (or double-knockout) mice of each strain with wild-type males. Under normal condition, each knockout strain showed normal implantation. The pregnant females were then subjected to E2 treatment (08:30, day 4) for 8 h, followed by uterine immunofluorescence detection of Aqp5/8 (16:30, day 4), intrauterine fluid volume measurement (16:30, day 4) and implantation site examination (08:30, day 5). Upon E2 treatment, both Aqp5−/− and Aqp8−/− mice showed significantly decreased luminal fluid volume compared with wild-type mice, and an even greater decrease was observed in Aqp5−/−Aqp8−/− uteri (Figure 1N). The number of implantation sites in Aqp5−/− and Aqp5−/−Aqp8−/− mice was also significantly increased compared with the wild type (Figure 1O and 1P). However, even in the Aqp5−/−Aqp8−/− uteri, the excess of luminal fluid was not completely prevented and the number of implantation sites was not fully restored, which is not surprising as E2 treatment also affects the expression of other ion channels and genes involved in regulating uterine receptivity and embryo implantation15. Nonetheless, the substantial correction of excessive luminal fluid retention and the increase in implantation rates in mice lacking Aqp5/8 suggest that these two aquaporins are responsible for E2-induced abnormal luminal fluid accumulation.Previous reports have shown that a decreased ratio of P4/E2 is a predictor of human implantation failure, and P4 supplementation could neutralize the adverse effects of elevated E2 level in mouse model4. Similarly, improved implantation rate caused by P4 treatment was confirmed in our experimental system (Figure 1Q), and we further demonstrated that co-injection of P4 along with E2 significantly decreased E2-induced upregulation of Aqp5 and Aqp8 expression, and reduced the excessive luminal fluid volume (Figure 1Q), which in our belief, is a major contributing factor to the improved implantation.In summary, the present study demonstrates a novel mechanism that Aqp5/8-dependent excessive intrauterine fluid accumulation is a major contributor to the supraphysiological level of E2-induced aberrant embryo implantation, which represents a critical cause of implantation failure aside from the well-recognized factors of endometrial receptivity. The driving force behind the excessive water movement is an important issue that warrants further investigation. Our data also showed that manipulation of the P4/E2 ratio could improve implantation rate through controlling uterine Aqp5/8-dependent intrauterine fluid accumulation, thus providing a molecular basis for improving IVF-ET outcomes.This work was supported by the National Basic Research Program of China (2011CB944400, 2011CB710905, 2012CB944702 and 2015CB943000), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA04020202-20 and XDA04020419) and the National Natural Science Foundation of China (31200879 and 31300957).(Supplementary information is linked to the online version of the paper on the Cell Research website.)

Multipotent skin-derived precursors (SKPs) are dermal stem cells with the capacity to reconstitute the dermis and other tissues, such as muscles and the nervous system. Thus, the easily available human SKPs (hSKPs) hold great promises in regenerative medicine. However, long-term expansion is difficult for hSKPs in vitro. We previously demonstrated that hSKPs senesced quickly under routine culture conditions. To identify the underlying mechanisms so as to find an effective way to expand hSKPs, time-dependent microarray analysis of gene expression in hSKPs during in vitro culture was performed. We found that the senescence of hSKPs had a unique gene expression pattern that differs from reported typical senescence. Subsequent investigation ruled out the role of DNA damage and classical p53 and p16INK4a signaling in hSKP senescence. Examination of cyclin-dependent kinase inhibitors revealed the involvement of p15INK4b and p27KIP1. Further exploration about upstream signals indicated the contribution of Akt hypo-activity and FOXO3 to hSKP senescence. Forced activation of Akt and knockdown of FOXO3, p15INK4b and p27KIP1 effectively inhibited hSKP senescence and promoted hSKP proliferation. The unique senescent phenotype of human dermal stem cells and the role of Akt-FOXO3-p27KIP1/p15INK4b signaling in regulating hSKP senescence provide novel insights into the senescence and self-renewal regulation of adult stem cells. The present study also points out a way to propagate hSKPs in vitro so as to fulfill their promises in regenerative medicine.

Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in hypergravity conditions does not affect the normal development and actin filament structures of mouse embryos.

Coordinated uterine-embryonic axis formation and decidual remodeling are hallmarks of mammalian post-implantation embryo development. Embryonic-uterine orientation is determined at initial implantation and synchronized with decidual development. However, the molecular mechanisms controlling these events remain elusive despite its discovery a long time ago. In the present study, we found that uterine-specific deletion of Rbpj, the nuclear transducer of Notch signaling, resulted in abnormal embryonic-uterine orientation and decidual patterning at post-implantation stages, leading to substantial embryo loss. We further revealed that prior to embryo attachment, Rbpj confers on-time uterine lumen shape transformation via physically interacting with uterine estrogen receptor (ERα) in a Notch pathway-independent manner, which is essential for the initial establishment of embryo orientation in alignment with uterine axis. While at post-implantation stages, Rbpj directly regulates the expression of uterine matrix metalloproteinase in a Notch pathway-dependent manner, which is required for normal post-implantation decidual remodeling. These results demonstrate that uterine Rbpj is essential for normal embryo development via instructing the initial embryonic-uterine orientation and ensuring normal decidual patterning in a stage-specific manner. Our data also substantiate the concept that normal mammalian embryonic-uterine orientation requires proper guidance from developmentally controlled uterine signaling.

Recently, physical factors in the local cellular microenvironment have been confirmed with strong influences on regulating stem cell fate. Despite the recent identification of the rotary cell culture system (RCCS) as a bioreactor for culturing stem cells, the underlying biological role provided by RCCS in the lineage differentiation of embryonic stem cells (ESCs) remains largely undefined. Here, we explored the embryoid body (EB) formation and subsequent differentiation of mouse ESCs in RCCS. We demonstrated that EBs formed in RCCS were more homogeneous and bigger in size compared with those in the static condition. Further, we determined that mesendoderm differentiation was prominently enhanced, while neuroectodermal differentiation was significantly suppressed in RCCS. Surprisingly, we found that Wnt/β-catenin signaling was greatly enhanced mainly due to the increased expression of Wnt3 during ESC differentiation in RCCS. Inhibition of Wnt/β-catenin signaling by DKK1 decreased the expression of Brachyury and attenuated mesendoderm differentiation in RCCS. Intriguingly, Wnt3a markedly increased Brachyury expression under static condition rather than in RCCS. Taken together, our findings uncover a new role of rotary suspension culture in initializing the early differentiation of ESCs.

The discovery of sperm-borne RNAs (mRNAs and small non-coding RNAs) has opened the possibility of additional paternal contributions aside from providing the DNA1. It has been reported that the incoming sperm can provide information for its host egg cytoplasm, which functionally influences the order of cell division2, possibly via delivering RNAs. Indeed, the sperm-borne miRNA and mRNA have been demonstrated as active players in early embryo development3 and transgenerational epigenetic inheritance4. However, given the diversity of small RNA classes (miRNA, endo-siRNA, piRNA, etc.) generated during spermatogenesis, the contents and profiles of the small RNA population carried by mature sperm remain undefined. In the present study, we isolated mature sperm from the cauda epididymis of adult male mice (Supplementary information, Data S1). The purity of sperm was > 99% as evaluated by microscopy and was confirmed by RT-PCR analyses of different biomarkers (Supplementary information, Figure S1A and S1B). The RNA extracted from mature sperm, adult testis, and uterus were processed for small RNA (< 40 nt) deep sequencing (Supplementary information, Figure S1C, S1D, S1E and Data S1). The total small RNA reads and genome-mapping statistic data (Supplementary information, Table S1) showed an abundance of small RNAs carried by mature sperm. The overall length distribution of small RNAs (Figure 1A) revealed that the dominant reads from mature sperm were at 29-34 nt, slightly different from adult testis (26-32 nt), and distinct from uterus (21-23 nt). The majority of the 26-32 nt small RNAs in mouse testis are piwi-interacting RNAs (piRNAs), which are actively involved in retrotransposon silencing that protects the integrity of the genome5. As it was initially suggested that piRNAs are absent in the cauda epididymis6 and that mammalian PIWI proteins (MILI, MIWI, MIWI2) are not expressed in mature sperm5, the abundant existence of 29-34 nt small RNAs in the mature sperm is somewhat surprising to us and suggests that they might be different from the well-known piRNA population from testis. Further analysis has revealed a distinct signature for these mature-sperm-enriched small RNAs, which represent a novel class of abundantly expressed small RNAs that can be grouped into distinct families. The small RNAs within each family showed identical 5′ sequences and only differed at their distal 3′ ends (Figure 1B and Supplementary information, Figure S2), suggesting that they are derived from the same precursor sequence. Particularly, two of these small RNA families were extremely enriched, which comprised 38.19% (family-1) and 19.14% (family-2) of all small RNA reads, respectively (Figure 1B), and together they accounted for the majority of the 30-34 nt small RNA population (Figure 1C).To further characterize these mature-sperm-enriched small RNAs, we performed BLAT searches for the top two abundant families against the mouse genomic databases (mm9). As shown in Figure 1D, these small RNAs are located at multiple sites on the genome, with several clusters on chromosomes 1, 8 and 13. Most strikingly, each of these genomic locations corresponds to a tRNA locus (Figure 1D). By further comparing with the genomic tRNA database, we found that each of these small RNA families unanimously matches to the 5′ half of a specific tRNA, with cleavage sites located preferentially at the anticodon loop (30-34 nt from the 5′ end), as illustrated for families 1-2 (Figure 1E and 1F) and for families 3-7 (Supplementary information, Figure S2). Their ultra-high enrichment (Figure 1G) and the preferential cleavage sites and length distributions strongly suggested that these small RNAs are not generated randomly by tRNA degradation, but under strict cleavage regulations. As these small RNAs are highly enriched in mature sperm and are derived from tRNAs, we termed them “mature-sperm-enriched tRNA-derived small RNAs” (mse-tsRNAs).Indeed, recent evidence has demonstrated that tRNA-derived RNA fragments are biologically functional7, and their production could be induced under various stress conditions (physical or chemical stress) by specific RNase8. The mature sperm is produced from testicular spermatogenesis followed by maturation during passage through the epididymis. The physiological condition and specific enzyme governing mse-tsRNAs production and accumulation in mature sperm are currently unknown.To monitor the biogenesis of mse-tsRNAs during sperm formation, we next analyzed purified mouse testicular spermatogenic cells (type A spermatogonia, pachytene spermatocytes, round spermatids and elongated spermatids) and mature sperm. We found that the levels of mse-tsRNA family-1 and -2 were relatively constant during the early stages of spermatogenesis, and were substantially increased at late- (family-1) or post-spermatogenesis (family-2) (Figure 1H). The RT-PCR results were further confirmed by analyzing small RNA deep-sequencing data obtained from purified mouse testicular spermatogenic cells9 and mature sperm (Figure 1I and 1J). By analyzing the PCR product size followed by product sequencing, we could find both mse-tsRNAs and their tRNA precursors, supporting the hypothesis that the mse-tsRNAs are derived from tRNA cleavage (Figure 1H). Interestingly, we consistently observed a sequence (52 nt) in mse-tsRNA family-2 PCR products, which is shorter than the expected length of intact tRNA and mapped to its 5′ portion (Figure 1H), suggesting the involvement of a two-step cleavage of tRNA in generating mse-tsRNA family-2. The increase of mse-tsRNA family-1 and -2 seems not to correlate with the expression of mammalian PIWI proteins (MILI, MIWI, MIWI2)5, suggesting that they might not be closely related to the piRNAs, and their ultra-high enrichment in mature sperm might be due to specific tRNA cleavage and/or selective accumulation of cleavage products at late- or post-spermatogenesis (such as during epididymal transition). The underlying mechanisms are currently unknown.We also analyzed the relative expression of miR-34c by RT-PCR as a quality control, as its expression in spermatogenic cells and mature sperm have been previously reported3,10. As shown in Figure 1H, our results were consistent with previous reports that miR-34c was almost absent in spermatogonia, but was highly expressed from pachytene spermatocytes and continued to be highly expressed in spermatids10 and mature sperm3. Note that miR-34c expression in mature sperm is much less than that of mse-tsRNA family-1 and -2, as shown by the RT-PCR results and by the reads number from our miRNA profiling database (Supplementary information, Figure S3).It is important to determine whether the sperm-borne mse-tsRNAs are located in sperm head, which could indicate their potential delivery into oocytes at fertilization. Using established methods to isolate purified sperm heads, we demonstrated that mse-tsRNAs are abundantly localized in the purified sperm head (Figure 1K and 1L), suggesting that they could be delivered into oocytes at fertilization. We next analyzed the expression levels of mse-tsRNA family-1 in oocytes, zygotes and parthenogenetically activated oocytes. Surprisingly, quantitative RT-PCR analysis revealed that the level of mse-tsRNA family-1 in zygotes is significantly lower than that in the oocytes and parthenogenetically activated oocytes (Supplementary information, Figure S4). These results might suggest a fertilization-triggered usage/consumption of mse-tsRNAs, which may reflect a functional role for mse-tsRNAs in early embryo events.As the mse-tsRNAs are derived from their tRNA templates, their sequence conservation could simply reflect the evolutionary conservation of their tRNA precursors. As shown in the evolutionary conservation analysis (Figure 1M and Supplementary information, Table S2), the tRNAs generating mse-tsRNA families 1-7 are highly conserved in vertebrate species, from fish to mammals, but absent from the worms, flies and plants. Indeed, besides our reported data for mouse sperm, existing small RNA datasets have shown that the mse-tsRNA family-1 is among the most highly expressed small RNA sequences in zebra fish testis (NCBI GEO Datasets: GSM830247)11 and human sperm (NCBI GEO Datasets: GSM530235)12, supporting the spermatozoal expression of mse-tsRNAs in a wide range of species. The expression of mse-tsRNA family-1 and -2 in mature sperm from mouse, rat and human were further analyzed using RT-PCR and confirmed by product sequencing (Figure 1N). These results suggest that mse-tsRNAs might serve as an ancient paternal element with evolutionarily conserved functions.Taken together, the present study revealed a previously hidden layer of sperm-borne small RNAs, identifying a novel class of tRNA-derived mse-tsRNAs with ultra-high accumulation in mature sperm. The biogenesis and function of these mse-tsRNAs are interesting topics that warrant future investigations.This research was supported by National Basic Research Program of China (2011CB944401 and 2011CB710905), Strategic Priority Research Program of the Chinese Academy of Sciences (XDA 01010202), National Natural Science Foundation of China (31200879), and Sciences Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-R-06).(Supplementary information is linked to the online version of the paper on the Cell Research website.)

The uterus is an indispensable organ for the development of a new life in eutherian mammals. The female mammalian reproductive capacity diminishes with age. In this respect, the senescence of uterine endometrium is convinced to contribute to this failure. This review focuses on the physiological function of the uterus and the related influence of aging mainly in rodent models. A better understanding of the underlying mechanisms governing the process of uterine aging is hoped to generate new strategies to prolong the reproductive lifespan in humans.

In the journey from the male to female reproductive tract, mammalian sperm experience a natural osmotic decrease (e.g., in mouse, from ~415 mOsm in the cauda epididymis to ~310 mOsm in the uterine cavity). Sperm have evolved to utilize this hypotonic exposure for motility activation, meanwhile efficiently silence the negative impact of hypotonic cell swelling. Previous physiological and pharmacological studies have shown that ion channel-controlled water influx/efflux is actively involved in the process of sperm volume regulation; however, no specific sperm proteins have been found responsible for this rapid osmoadaptation. Here, we report that aquaporin3 (AQP3) is a sperm water channel in mice and humans. Aqp3-deficient sperm show normal motility activation in response to hypotonicity but display increased vulnerability to hypotonic cell swelling, characterized by increased tail bending after entering uterus. The sperm defect is a result of impaired sperm volume regulation and progressive cell swelling in response to physiological hypotonic stress during male-female reproductive tract transition. Time-lapse imaging revealed that the cell volume expansion begins at cytoplasmic droplet, forcing the tail to angulate and form a hairpin-like structure due to mechanical membrane stretch. The tail deformation hampered sperm migration into oviduct, resulting in impaired fertilization and reduced male fertility. These data suggest AQP3 as an essential membrane pathway for sperm regulatory volume decrease (RVD) that balances the “trade-off” between sperm motility and cell swelling upon physiological hypotonicity, thereby optimizing postcopulatory sperm behavior.

Upon ejaculation, mammalian sperm experience a natural osmotic decrease during male to female reproductive tract transition. This hypo-osmotic exposure not only activates sperm motility, but also poses potential harm to sperm structure and function by inducing unwanted cell swelling. In this physiological context, regulatory volume decrease (RVD) is the major mechanism that protects cells from detrimental swelling, and is essential to sperm survival and normal function. Aquaporins are selective water channels that enable rapid water transport across cell membranes. Aquaporins have been implicated in sperm osmoregulation. Recent discoveries show that Aquaporin-3 (AQP3), a water channel protein, is localized in sperm tail membranes and that AQP3 mutant sperm show defects in volume regulation and excessive cell swelling upon physiological hypotonic stress in the female reproductive tract, thereby highlighting the importance of AQP3 in the postcopulatory sperm RVD process. In this paper, we discuss current knowledge, remaining questions and hypotheses about the function and mechanismic basis of aquaporins for volume regulation in sperm and other cell types.

In mouse, decidualization is characterized by the proliferation of stromal cells and their differentiation into specialized type of cells (decidual cells) with polyploidy, surrounding the implanting blastocyst. However, the mechanisms involved in these processes remain poorly understood. Using multiple approaches, we have examined the role of Adam12 in decidualization during early pregnancy in mice. Adam12 is spatiotemporally expressed in decidualizing stromal cells in intact pregnant females and in pseudopregnant mice undergoing artificially induced decidualization. In the ovariectomized mouse uterus, the expression of Adam12 is upregulated after progesterone treatment, which is primarily mediated by nuclear progesterone receptor. In a stromal cell culture model, the expression of Adam12 gradually rises with the progression of stromal decidualization, whereas the attenuated expression of Adam12 after siRNA knockdown significantly blocks the progression of decidualization. Our study suggests that Adam12 is involved in promoting uterine decidualization during pregnancy.

The enrichment and identification of human epidermal stem cells (EpSCs) are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rapidly adherent keratinocytes into three populations that range in size from 5–7 m (population A), to 7–9 m (population B), to 9 m (population C) in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of 1-integrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency (CFE). When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to 1-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.

Recently, the study on “induced pluripotent stem cells” (iPS cells) has made a great breakthrough, and it is considered as a new milestone in the history of life science. This progress has updated our traditional concepts about pluripotency control, and provided people with a brand-new strategy for somatic cell nuclear reprogramming. In virtue of its availability and stability, this method holds great potential in both biological and clinical research. In order to introduce this rising field of study, this paper starts with an overview of the development of iPS cell establishment, describes the key steps in generating iPS cells, elaborates several relevant scientific issues, and evaluates its current restrictions and promises in future research.

The developmental capacity of mouse embryos in the Chinese SJ-8 Satellite was observed by real time micrography and telecontrol image transmission. Frozen/thawed 4-cell embryos and blastocysts injected with mouse epidemical stem cells were placed in a specially sealed embryonic incubator, and then the incubator was loaded in a space embryonic culture box devised for space-flight. After the satellite launched and arrived at the anticipated orbit, the real time micrography device was opened based on the telecontrol operational technology. Real time micrographs of the mouse embryos were obtained and stored every 3 hours, then the data of images were transmitted at the suitable time. The experiment persisted for 72 hours. The results showed that during space-flight, most mouse embryos cultured in the sealed culture unit kept integrity and natural structure, their location had minor change, but the embryos did not develop. However, the experiment performed on the ground in the same device showed that 4-cell mouse embryos could develop to blastocysts and hatched blastocysts. It may be concluded that the space environment, especially the change of gravity was likely to harm development of the mouse embryo.

The distribution of intrauterine embryo implantation site(s) in most mammalian species shows remarkably constant patterns: in monotocous species such as humans, an embryo tends to implant in the uterine fundus; in polytocous species such as rodents, embryos implant evenly along the uterine horns. These long-time evolved patterns bear great biological significance because disruption of these patterns can have adverse effects on pregnancies. However, lack of suitable models and in vivo monitoring techniques has impeded the progress in understanding the mechanisms of intrauterine embryo distribution. These obstacles are being overcome by genetically engineered mouse models and newly developed high-resolution ultrasound. It has been revealed that intrauterine embryo distribution involves multiple events including uterine sensing of an embryo, fine-tuned uterine peristaltic movements, time-controlled uterine fluid reabsorption and uterine luminal closure, as well as embryo orientation. Diverse molecular factors, such as steroid hormone signaling, lipid signaling, adrenergic signaling, developmental genes, ion/water channels, and potentially embryonic signaling are actively involved in intrauterine embryo distribution. This review covers the biomechanical and molecular aspects of intrauterine embryo distribution (embryo spacing at the longitudinal axis and embryo orientation at the vertical axis), as well as its pathophysiological roles in human reproductive medicine. Future progress requires multi-disciplinary research efforts that will integrate in vivo animal models, clinical cases, physiologically relevant in vitro models, and biomechanical/computational modeling. Understanding the mechanisms for intrauterine embryo distribution could potentially lead to development of therapeutics for treating related conditions in reproductive medicine.

Microgravity was simulated with a rotating wall vessel bioreactor (RWVB) in order to study its effect on pre-implantation embryonic development in mice. Three experimental groups were used: stationary control, rotational control and clinostat rotation. Three experiments were performed as follows. The first experiment showed that compared with the other two (control) groups, embryonic development was significantly retarded after 72 h in the clinostat rotation group. The second experiment showed that more nitric oxide (NO) was produced in the culture medium in the clinostat rotation group after 72 h (P < 0.05), and the nitric oxide synthase (NOS) activity in this group was significantly higher than in the controls (P < 0.01). In the third experiment, we studied apoptosis in the pre-implantation mouse embryos after 72 h in culture and found that Annexin-V staining was negative in the normal (stationary and rotational control) embryos, but the developmentally retarded (clinostat rotation) embryos showed a strong green fluorescence. These results indicate that microgravity induced developmental retardation and cell apoptosis in the mouse embryos. We presume that these effects are related to the higher concentration of NO in the embryos under microgravity, which have cause cytotoxic consequences.