Within the seminiferous tubules there are two major cell types, namely male germ cells and Sertoli cells. Recent studies have demonstrated that male germ cells and Sertoli cells can have significant applications in treating male infertility and other diseases. However, primary male germ cells are hard to proliferate in vitro and the number of spermatogonial stem cells is scarce. Therefore, methods that promote the expansion of these cell populations are essential for their use from the bench to the bed side. Notably, a number of cell lines for rodent spermatogonia, spermatocytes and Sertoli cells have been developed, and significantly we have successfully established a human spermatogonial stem cell line with an unlimited proliferation potential and no tumor formation. This newly developed cell line could provide an abundant source of cells for uncovering molecular mechanisms underlying human spermatogenesis and for their utilization in the field of reproductive and regenerative medicine. In this review, we discuss the methods for establishing spermatogonial, spermatocyte and Sertoli cell lines using various kinds of approaches, including spontaneity, transgenic animals with oncogenes, simian virus 40 (SV40) large T antigen, the gene coding for a temperature-sensitive mutant of p53, telomerase reverse gene (Tert), and the specific promoter-based selection strategy. We further highlight the essential applications of these cell lines in basic research and translation medicine.
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Hong Wang, Liping Wen, Qingqing Yuan, Min Sun, Minghui Niu, and Zuping He
Tong Sun, Shi-Jie Li, Hong-Lu Diao, Chun-Bo Teng, Hong-Bin Wang, and Zeng-Ming Yang
Cyclooxygenase (COX), a rate-limiting enzyme that produces prostaglandins (PGs) from arachidonic acid, exists in two isoforms, COX-1 and COX-2. PGE2 synthase (PGES) is a terminal prostanoid synthase and can enzymatically convert the cyclooxygenase product PGH2 to PGE2, including two isoforms: microsomal PGES (mPGES) and cytosolic PGES (cPGES). cPGES is predominantly linked with COX-1 to promote the immediate response. mPGES is preferentially coupled with the inducible COX-2 to promote delayed PGE2 generation. COX-2-deficient female mice are infertile with abnormalities in ovulation, fertilization, implantation and decidualization. The aim of this study was to examine immunohistochemically the expression pattern of COX-1, COX-2, mPGES and cPGES proteins in the endometrium of the rhesus monkey during the menstrual cycle. COX-1 immunostaining was mainly localized in the luminal epithelium and glandular epithelium near the lumen, and detected in all the stages during the menstrual cycle. COX-2 immunostaining was mainly localized in the luminal and glandular epithelium, and strongly shown during the mid-luteal phase (days 16 and 20) of the menstrual cycle. There was a strong cPGES immunostaining in the luminal and glandular epithelium on days 12, 16, 20 and 25 of the menstrual cycle. mPGES immunostaining was strongly detected in the glandular epithelium on days 20 and 25 of the menstrual cycle. These data suggest that the coupling of cPGES and COX-1 in the luminal epithelium may be responsible for the synthesis of PGE2 in monkey endometrium, and the coupling of mPGES and COX-2 in the glandular epithelium may be of importance for preparing the receptive endometrium.
Jun-Zuo Wang, Hong-Shu Sui, De-Qiang Miao, Na Liu, Ping Zhou, Li Ge, and Jing-He Tan
The objectives of this study were to investigate the effect of heat stress during in vitro maturation on the developmental potential of mouse oocytes and to determine whether the deleterious effect was on the nuclear or cytoplasmic component. While rates of oocyte nuclear maturation (development to the metaphase II stage) did not differ from 37 to 40 °C, rates for blastocyst formation decreased significantly as maturation temperature increased from 38.5 to 39 °C. Chromosome spindle exchange showed that while blastocyst formation did not differ when spindles matured in vivo or in vitro at 37, 40 or 40.7 °C were transplanted into in vivo matured cytoplasts, no blastocyst formation was observed when in vivo spindles were transferred into the 40 °C cytoplasts. While oocytes reconstructed between 37 °C ooplasts and 37 or 40 °C karyoplasts developed into 4-cell embryos at a similar rate, no oocytes reconstituted between 40 °C ooplasts and 37 °C spindles developed to the 4-cell stage. Immunofluorescence microscopy revealed impaired migration of cortical granules and mitochondria in oocytes matured at 40 °C compared with oocytes matured at 37 °C. A decreased glutathione/GSSG ratio was also observed in oocytes matured at 40 °C. While spindle assembling was normal and no MAD2 was activated in oocytes matured at 37 or 40 °C, spindle assembling was affected and MAD2 was activated in some of the oocytes matured at 40.7 °C. It is concluded that 1) oocyte cytoplasmic maturation is more susceptible to heat stress than nuclear maturation, and 2) cytoplasmic rather than nuclear components determine the pre-implantation developmental capacity of an oocyte.
Xing Su, Yi Hu, Ying Li, Jing-Li Cao, Xue-Qin Wang, Xu Ma, and Hong-Fei Xia
Although the relationship between polymorphisms in microRNAs (miRNAs) and recurrent pregnancy loss (RPL) has been studied, there is very little data available in the literature. In the present study, we scanned 55 potentially functional polymorphisms in the miRNA coding region in Chinese women with unexplained RPL (URPL; no. 2011-10). The rs6505162 C>A in the MIR423 coding region was found to be significantly associated with the occurrence of human URPL. The rare A allele contributed to an increase in the expression of mature MIR423. C to A substitution in the polymorphism rs6505162 in pre-MIR423 repressed cell proliferation and migratory capacity. Further investigations showed that MIR 423 could inversely regulate the expression of proliferation-associated 2 group 4 (PA2G4) by binding the 3′-UTR of PA2G4. Dual-luciferase assay indicated that the A allele in the polymorphism rs6505162 could more effectively suppress the expression of PA2G4 than the C allele could. Collectively, the present data suggest that rs6505162 C>A in pre-MIR423 may contribute to the genetic predisposition to RPL by disrupting the production of mature MIR42 3 and its target gene, which consequently interferes with MIR423 functioning.
Hsien-Ming Wu, Hsin-Shih Wang, Hong-Yuan Huang, Yung-Kuei Soong, Colin D MacCalman, and Peter C K Leung
Type I GnRH (GnRH-I, GNRH1) and type II GnRH (GnRH-II, GNRH2), each encoded by separate genes, have been identified in humans. The tissue distribution and functional regulation of GnRH-I and GnRH-II clearly differ despite their comparable cDNA and genomic structures. These hormones exert their effects by binding to cell surface transmembrane G protein coupled receptors and stimulating the Gq/11 subfamily of G proteins. The hypothalamus and pituitary are the main origin and target sites of GnRH, but numerous studies have demonstrated that extra-hypothalamic GnRH and extra-pituitary GnRH receptors exist in different reproductive tissues such as the ovary, endometrium, placenta, and endometrial cancer cells. In addition to endocrine regulation, GnRH is also known to act in an autocrine and paracrine manner to suppress cell proliferation and activate apoptosis in the endometrium and endometrial cancer cells through several mechanisms. Both GnRH-I and GnRH-II exhibit regulatory roles in tissue remodelling during embryo implantation and placentation, which suggests that these hormones may have important roles in embryo implantation and early pregnancy. The presence of varied GnRH and GnRH receptor systems demonstrate their different roles in distinct tissues using dissimilar mechanisms. These may result in the generation of new GnRH analogues used for several hormone-related diseases.
Wenjun Wang, Hong Chen, Ruiqi Li, Nengyong Ouyang, Jinghua Chen, Lili Huang, Meiqi Mai, Ningfeng Zhang, Qingxue Zhang, and Dongzi Yang
Our previous study has demonstrated that luteinized granulosa cells (GCs) have the potential to proliferate and that the telomerase activity (TA) of luteinized GCs may predict the clinical outcomes of IVF treatment. However, in the field of telomere research, there have always been different opinions regarding the significance of TA and telomere length (TL). Thus, in the present study, we compared the effects of these two parameters on IVF treatment outcomes in the same individuals. TL did not differ significantly between the pregnant group and the non-pregnant group. The TA, number of retrieved oocytes and rate of blastocyst transfer were significantly higher in the pregnant group than in the non-pregnant group (0.8825 OD×mm, 12.75±2.20 and 34.48%, respectively, in the pregnant group vs 0.513 OD×mm, 11.60±0.93 and 14.89%, respectively, in the non-pregnant group (P<0.05)), while basal FSH level was lower in the pregnant group than in the non-pregnant group. The subjects did not differ with regard to ovarian stimulation or other clinical characteristics. A TA increase of 1 OD×mm increased the chance of becoming pregnant 4.769-fold (odds ratio: 5.769, 95% CI: 1.434–23.212, P<0.014). The areas under the receiver operating characteristic curves were 0.576 for TL and 0.674 for TA (P=0.271 and P<0. 012 respectively). The corresponding cut-off points were 4.470 for TL and 0.650 OD×mm for TA. These results demonstrate that TA is a better predictor of pregnancy outcomes following IVF treatment than TL. No other clinical parameters, including age, baseline FSH level or peak oestradiol level, distinguished between the pregnant group and the non-pregnant group as effectively as TA.
Hua-Yu Lian, Yan Gao, Guang-Zhong Jiao, Ming-Ju Sun, Xiu-Fen Wu, Tian-Yang Wang, Hong Li, and Jing-He Tan
In this study, using a mouse model, we tested the hypothesis that restraint stress would impair the developmental potential of oocytes by causing oxidative stress and that antioxidant supplementation could overcome the adverse effect of stress-induced oxidative stress. Female mice were subjected to restraint stress for 24 h starting 24 h after equine chorionic gonadotropin injection. At the end of stress exposure, mice were either killed to recover oocytes for in vitro maturation (IVM) or injected with human chorionic gonadotropin and caged with male mice to observe in vivo development. The effect of antioxidants was tested in vitro by adding them to IVM medium or in vivo by maternal injection immediately before restraint stress exposure. Assays carried out to determine total oxidant and antioxidant status, oxidative stress index, and reactive oxygen species (ROS) and glutathione levels indicated that restraint stress increased oxidative stress in mouse serum, ovaries, and oocytes. Whereas the percentage of blastocysts and number of cells per blastocyst decreased significantly in oocytes from restraint-stressed mice, addition of antioxidants to IVM medium significantly improved their blastocyst development. Supplementation of cystine and cysteamine to IVM medium reduced ROS levels and aneuploidy while increasing glutathione synthesis and improving pre- and postimplantation development of oocytes from restraint-stressed mice. Furthermore, injection of the antioxidant epigallocatechin gallate into restraint-stressed mice significantly improved the blastocyst formation and postimplantation development of their oocytes. In conclusion, restraint stress at the oocyte prematuration stage impaired the developmental potential of oocytes by increasing oxidative stress and addition of antioxidants to IVM medium or maternal antioxidant injection overcame the detrimental effect of stress-induced oxidative stress. The data reported herein are helpful when making attempts to increase the chances of a successful outcome in human IVF, because restraint was applied at a stage similar to the FSH stimulation period in a human IVF program.
Na Liu, Yan-Guang Wu, Guo-Cheng Lan, Hong-Shu Sui, Li Ge, Jun-Zuo Wang, Yong Liu, Tian-Wu Qiao, and Jing-He Tan
Inhibiting oocyte aging is important not only for healthy reproduction but also for the success of assisted reproduction techniques. Although our previous studies showed that cumulus cells accelerated aging of mouse oocytes, the underlying mechanism is unknown. The objective of this paper was to study the effects of pyruvate and cumulus cells on mouse oocyte aging. Freshly ovulated mouse cumulus–oocyte complexes (COCs) or cumulus-denuded oocytes (DOs) were cultured in Chatot-Ziomek-Bavister (CZB) medium or COC-conditioned CZB medium supplemented with different concentrations of pyruvate before being examined for aging signs and developmental potential. Pyruvate supplementation to CZB medium decreased rates of ethanol-induced activation in both COCs and DOs by maintaining their maturation-promoting factor activities, but more pyruvate was needed for COCs than for DOs. Addition of pyruvate to the COC-conditioned CZB also alleviated aging of DOs. Observations on cortical granules, level of BCL2 proteins, histone acetylation, intracellular concentration of glutathione, and embryo development all confirmed that pyruvate supplementation inhibited aging of mouse oocytes. It is concluded that the aging of mouse oocytes, facilitated by culture in COCs, can be partially prevented by the addition of pyruvate to the culture medium.
Yan Sun, Yifen Yang, Ziran Jiang, Feiyu wang, Kun Han, Linjun Hong, Jianhua Cao, and Mei Yu
Transforming the endometrial luminal epithelium (LE) into a receptive state is a requisite event for successful embryo implantation. This study suggests the role of a transcription factor in regulating endometrial LE receptivity.
The endometrial luminal epithelium (LE) undergoes extensive remodeling during implantation to establish receptivity of the uterus in response to the conceptus signals, such as interleukin 1β (IL1B). But the mechanisms remain to be fully understood. This study investigated the role of CCAAT/enhancer-binding protein β (C/EBP-β) in regulating pig endometrial LE receptivity. Our results showed that C/EBP-β was expressed and activated only in the endometrial LE in an implantation-dependent manner. In addition, C/EBP-β was highly activated at the pre-attachment stage compared to the attachment stage, and its activation was correlated with the expression of IL1B-dependent extracellular signal-regulated kinases1/2-p90 ribosomal S6 kinase signaling axis. Subsequent chromatin immunoprecipitation (ChIP)-sequencing analysis revealed that the binding of C/EBP-β within the promoter was positively associated with the transcription of genes related to cell remodeling. One such gene is matrix metalloproteinase 8 (MMP8), which is responsible for extracellular matrix degradation. The expression of MMP8 was abundant at the pre-attachment stage but dramatically declined at the attachment stage in the endometrial LE. Consistent with C/EBP-β, the expression and activation of MMP8 were limited to the endometrial LE in an implantation-dependent manner. Using ChIP-qPCR and electrophoresis mobility shift assay approaches, we demonstrated that C/EBP-β regulated the expression of the MMP8 gene during implantation. Furthermore, we detected that MMP8 and one of its substrates, type II collagen, showed a mutually exclusive expression pattern in pig endometrial LE during implantation. Our findings indicate that C/EBP-β plays a role in pig endometrial LE receptivity by regulating cell remodeling-related genes, such as MMP8, in response to conceptus signals during implantation.
Songcun Wang, Fengrun Sun, Mutian Han, Yinghua Liu, Qinyan Zou, Fuxin Wang, Yu Tao, Dajin Li, Meirong Du, Hong Li, and Rui Zhu
There is delicate crosstalk between fetus-derived trophoblasts (Tros) and maternal cells during normal pregnancy. Dysfunctions in interaction are highly linked to some pregnancy complications, such as recurrent spontaneous abortion (RSA), pre-eclampsia and fetal growth restriction. Hyaluronan (HA), the most abundant component of extracellular matrix, has been reported to act as both a pro- and an anti-inflammatory molecule. Previously, we reported that HA promotes the invasion and proliferation of Tros by activating PI3K/Akt and MAPK/ERK1/2 signaling pathways. While lower HA secretion by Tros was observed during miscarriages than that during normal pregnancies, in the present study, we further confirmed that higher secretion of HA by Tros could induce M2 polarization of macrophages at the maternal–fetal interface by interacting with CD44 and activating the downstream PI3K/Akt-STAT-3/STAT-6 signaling pathways. Furthermore, HA could restore the production of IL-10 and other normal pregnancy markers by decidual macrophages (dMφs) from RSA. These findings underline the important roles of HA in regulating the function of dMφs and maintaining a normal pregnancy.