Quantitative proteomic analysis uncovers protein-expression profiles during gonadotropin-dependent folliculogenesis in mice†.

Ovarian follicle is the basic functional unit of female reproduction, and is composed of oocyte and surrounding granulosa cells. In mammals, folliculogenesis strictly rely on gonadotropin regulations to determine the ovulation and the quality of eggs. However, the dynamic changes of protein-expressing profiles in follicles at different developmental stages remain largely unknown. By performing mass-spectrometry-based quantitative proteomic analysis of mouse follicles, we provide a proteomic database (~3000 proteins) that covers three key stages of gonadotropin-dependent folliculogenesis. By combining bioinformatics analysis with in situ expression validation, we showed that our proteomic data well reflected physiological changes during folliculogenesis, which provided potential to predict unknown regulators of folliculogenesis. Additionally, by using the oocyte structural protein zona pellucida protein 2 as the internal control, we showed the possibility of our database to predict the expression dynamics of oocyte-expressing proteins during folliculogenesis. Taken together, we provide a high-coverage proteomic database to study protein-expression dynamics during gonadotropin-dependent folliculogenesis in mammals.

Asynchronous embryonic germ cell development leads to a heterogeneity of postnatal ovarian follicle activation and may influence the timing of puberty onset in mice.

BACKGROUND: Ovarian follicles, which are the basic units of female reproduction, are composed of oocytes and surrounding somatic (pre) granulosa cells (GCs). A recent study revealed that signaling in somatic preGCs controlled the activation (initial recruitment) of follicles in the adult ovaries, but it is also known that there are two waves of follicle with age-related heterogeneity in their developmental dynamics in mammals. Although this heterogeneity was proposed to be crucial for female reproduction, our understanding of how it arises and its significance is still elusive. RESULTS: In the current study, by deleting the key secreted factor KIT ligand from preGCs and analyzing the follicle cell developmental dynamics, we revealed distinct patterns of activation and growth associated with the two waves of follicles in mouse ovary. Our results confirmed that activation of adult wave follicles is initiated by somatic preGCs and dependent on the KIT ligand. By contrast, activation of first wave follicles, which are awakened from germ cells before follicle formation, can occur in the absence of preGC-secreted KIT ligand in postnatal ovaries and appears to be oocyte-initiated. We also found that the asynchronous activity of phosphatidylinositol 3 kinases (PI3K) signaling and meiotic process in embryonic germ cells lead to the follicle heterogeneity in postnatal ovaries. In addition, we supplied evidence that the time sequence of embryonic germ cell development and its related first wave follicle growth are correlated to the time of puberty onset in females. CONCLUSION: Taken together, our study provides evidence that asynchronous development of embryonic oocytes leads to the heterogeneity of postnatal ovarian follicle activation and development, and affects the timing of onset of puberty in females.

In vivo promotion of primordial follicle activation by stem cell factor treatment in mice with premature ovarian insufficiency and advanced age.

Dormant primordial follicles (PFs) are the most abundant reproductive resource in mammalian ovaries. With advances in the mechanism of study of the regulation of PF activation, PFs have been used to improve fertility in clinical practice. As a central controlling element of follicle activation signaling, the pre-granulosa cell-secreted stem cell factor (SCF; also known as KIT ligand, KITL), which initiates the growth of dormant oocytes, is an ideal natural activator that stimulates follicle activation. However, no systematic study has been conducted to identify the activating effect of SCF in vivo and in vitro. In this study, by combining an in vitro whole ovary culture system and several mouse models, we provide a series of experimental evidence that SCF is an efficient activator for improving PF activation in mouse ovaries. Our in vitro study showed that SCF increased phosphatidylinositol 3-kinase (PI3K) signaling and PF activation ratio in neonatal ovaries. In vivo ovarian non-invasive topical administrations of SCF to the ovaries efficiently improved follicle activation and development, oocyte retrieval ratio and fertility in inducible premature ovarian insufficiency mouse models and aged mice. Our study suggests that SCF is an efficient growth factor that can be applied to improve PF activation.

Synergistic effects of gene polymorphisms of the renin-angiotensin-aldosterone system on essential hypertension in Kazakhs in Xinjiang.

OBJECTIVE: To assess the synergistic effects of gene polymorphisms of the renin-angiotensin-aldosterone system (RAAS) on essential hypertension (EH) in Kazakhs in Xinjiang. METHODS: A cross-sectional case-control association study was conducted in 52 1 hypertensive and 623 normotensive subjects of Kazakh ethnicity on eight common single nucleotide polymorphisms (SNPs) interspersed over five genes of the RAAS. SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism. Interactions among the SNPs were analyzed by the multifactor dimensionality reduction method (MDR). RESULTS: In single-locus analysis, subjects with AGT -6G, ACE D, and CYP11B2 -344C had increased susceptibility to EH (OR: 1.249; 1.425; 1.201). When subgrouped by sex, males with the t allele of REN Taq I had decreased risk for EH (OR: 0.529), and those with AGT -6G and CYP11B2 -344 C had increased risk for EH (OR: 1.498; 1.449). In females, carrying ACE D increased the risk for EH. (OR: 1.327). In six AGT haplotypes, H1 was protective, while H3 increased susceptibility to EH (OR: 0.683; 2.025). Interaction analysis by MDR showed that there was a strong synergistic effect between ACE I/D and CY11B2 (T-344C) and a moderate interaction between both ACE I/D and CY11B2 T-344C and AGT A-6G. CONCLUSIONS: There was a strong synergistic effect between ACE I/D and CY11B2 T-344C and a moderate effect between both ACE I/D and CY11B2 T-344C and AGT A-6G. AGT -6G, ACE D, and CY11B2 -344C increased susceptibility to EH. REN Taq I, AGT -6G, CY11B2 -344 C and ACE D were associated with male and female EH, respectively. H1 and H3 of AGT were protective and risk haplotypes, respectively.

Synergistic effects of gene polymorphisms of the renin-angiotensin-aldosterone system on essential hypertension in Kazakhs in Xinjiang.

OBJECTIVE: To assess the synergistic effects of gene polymorphisms of the renin-angiotensin-aldosterone system (RAAS) on essential hypertension (EH) in Kazakhs in Xinjiang. METHODS: A cross-sectional case-control association study was conducted in 52 1 hypertensive and 623 normotensive subjects of Kazakh ethnicity on eight common single nucleotide polymorphisms (SNPs) interspersed over five genes of the RAAS. SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism. Interactions among the SNPs were analyzed by the multifactor dimensionality reduction method (MDR). RESULTS: In single-locus analysis, subjects with AGT -6G, ACE D, and CYP11B2 -344C had increased susceptibility to EH (OR: 1.249; 1.425; 1.201). When subgrouped by sex, males with the t allele of REN Taq I had decreased risk for EH (OR: 0.529), and those with AGT -6G and CYP11B2 -344 C had increased risk for EH (OR: 1.498; 1.449). In females, carrying ACE D increased the risk for EH. (OR: 1.327). In six AGT haplotypes, H1 was protective, while H3 increased susceptibility to EH (OR: 0.683; 2.025). Interaction analysis by MDR showed that there was a strong synergistic effect between ACE I/D and CY11B2 (T-344C) and a moderate interaction between both ACE I/D and CY11B2 T-344C and AGT A-6G. CONCLUSIONS: There was a strong synergistic effect between ACE I/D and CY11B2 T-344C and a moderate effect between both ACE I/D and CY11B2 T-344C and AGT A-6G. AGT -6G, ACE D, and CY11B2 -344C increased susceptibility to EH. REN Taq I, AGT -6G, CY11B2 -344 C and ACE D were associated with male and female EH, respectively. H1 and H3 of AGT were protective and risk haplotypes, respectively.

Lineage tracing of mutant granulosa cells reveals in vivo protective mechanisms that prevent granulosa cell tumorigenesis.

Ovarian granulosa cell tumors (GCTs) originate from granulosa cells (GCs) and represent the most common sex cord-stromal tumor in humans. However, the developmental regulations and molecular mechanisms underlying their etiology are largely unknown. In the current study, we combined a multi-fluorescent reporter mouse model with a conditional knockout mouse model, in which the tumor suppressor genes Pten and p27 were deleted in GCs, to perform cell lineage tracing of mutant GCs. We found that only 30% of ovaries with substantial mutant GCs developed into GCTs that derived from a single mutant GC. In-depth molecular analysis of the process of tumorigenesis demonstrated that up-regulation of immune evasion genes Cd24a and Cd47 led, in part, to the transition of mutant GCs to GCTs. Therefore, treatment with the Cd47 inhibitor RRX-001 was tested and found to efficiently suppress the growth of GCTs in vivo. Together, our study has revealed an immune evasion mechanism via CD24/CD47 upregulation to GCT formation, shedding light on the future potential clinical therapies for GCTs.

Analyzing the cellular and molecular atlas of ovarian mesenchymal cells provides a strategy against female reproductive aging.

Ovarian mesenchymal cells (oMCs) constitute a distinct microenvironment that supports folliculogenesis under physiological conditions. Supplementation of exogenous non-ovarian mesenchymal-related cells has been reported to be an efficient approach to improve ovarian functions. However, the development and cellular and molecular characteristics of endogenous oMCs remain largely unexplored. In this study, we surveyed the single-cell transcriptomic landscape to dissect the cellular and molecular changes associated with the aging of oMCs in mice. Our results showed that the oMCs were composed of five ovarian differentiated MC (odMC) populations and one ovarian mesenchymal progenitor (oMP) cell population. These cells could differentiate into various odMCs via an oMP-derived route to construct the ovarian stroma structures. Comparative analysis revealed that ovarian aging was associated with decreased quantity of oMP cells and reduced quality of odMCs. Based on the findings of bioinformatics analysis, we designed different strategies involving supplementation with young oMCs to examine their effects on female fertility and health. Our functional investigations revealed that oMCs supplementation prior to ovarian senescence was the optimal method to improve female fertility and extend the reproductive lifespan of aged females in the long-term.

Simulated microgravity reduces quality of ovarian follicles and oocytes by disrupting communications of follicle cells.

Ovarian follicles are the fundamental structures that support oocyte development, and communications between oocytes and follicle somatic cells are crucial for oogenesis. However, it is unknown that whether exposure to microgravity influences cellular communications and ovarian follicle development, which might be harmful for female fertility. By 3D culturing of ovarian follicles under simulated microgravity (SMG) conditions in a rotating cell culture system, we found that SMG treatment did not affect the survival or general growth of follicles but decreased the quality of cultured follicles released oocytes. Ultrastructure detections by high-resolution imaging showed that the development of cellular communicating structures, including granulosa cell transzonal projections and oocyte microvilli, were markedly disrupted. These abnormalities caused chaotic polarity of granulosa cells (GCs) and a decrease in oocyte-secreted factors, such as Growth Differentiation Factor 9 (GDF9), which led to decreased quality of oocytes in these follicles. Therefore, the quality of oocytes was dramatically improved by the supplementations of GDF9 and NADPH-oxidase inhibitor apocynin. Together, our results suggest that exposure to simulated microgravity impairs the ultrastructure of ovarian follicles. Such impairment may affect female fertility in space environment.

Imaging and tracing the pattern of adult ovarian angiogenesis implies a strategy against female reproductive aging.

Robust angiogenesis is continuously active in ovaries to remodel the ovary-body connections in mammals, but understanding of this unique process remains elusive. Here, we performed high-resolution, three-dimensional ovarian vascular imaging and traced the pattern of ovarian angiogenesis and vascular development in the long term. We found that angiogenesis was mainly active on ovarian follicles and corpus luteum and that robust angiogenesis constructs independent but temporary vascular networks for each follicle. Based on the pattern of ovarian angiogenesis, we designed an angiogenesis-blocking strategy by axitinib administration to young females, and we found that the temporary suppression of angiogenesis paused ovarian development and kept the ovarian reserve in the long term, leading to postponed ovarian senescence and an extension of the female reproductive life span. Together, by uncovering the detailed model of physiological ovarian angiogenesis, our experiments suggest a potential approach to delay female reproductive aging through the manipulation of angiogenesis.

Polycomb subunit Pcgf2 mediates ovulation and fertility through transcriptional regulation progesterone receptor.

Ovarian follicles are the fundamental structure to support oocyte development, which provides mature oocytes for offspring. This process requires granulosa cells (GCs) to respond to the midcycle surge of hormones, leading to GC proliferation and differentiation by a series of genes' transcriptional expression changes. Epigenetic mediator, Polycomb Repressive Complex 1 (PRC1) has been reported to function in fetal ovarian development. However, its functional relevance to folliculogenesis and ovulation remains unknown. In this study, we demonstrated that GC-selective depletion of PCGF2, a key component of PRC1, led to the loss of follicles, ovulation defects, and a lengthened estrus cycle, resulting in subfertility in female mice. The expression of PCGF2 is in the GCs of growing follicles and increases after human chorionic gonadotropin (hCG) stimulation. PCGF2 bound to the promoter of the key ovulation gene progesterone receptor (Pgr) and upregulated the expression of Pgr by targeting the epigenetic modification of H2AK119ub1 after hCG surge. Consistently, the expression of downstream genes of Pgr also sharply decreased, which resulted in the follicular rupture failed and oocyte entrapped in corpus luteum in GC-specific Pcgf2 knockout mice. Together, our study identified that PCGF2 is essential for folliculogenesis and ovulation via modulating hormone receptor expression.

Oocyte-derived microvilli control female fertility by optimizing ovarian follicle selection in mice.

Crosstalk between oocytes and surrounding somatic cells is crucial for mammalian oogenesis, but the structural mechanisms on oocytes to control female reproduction remain unknown. Here we combine endogenous-fluorescent tracing mouse models with a high-resolution live-cell imaging system to characterize oocyte-derived mushroom-like microvilli (Oo-Mvi), which mediate germ-somatic communication in mice. We perform 3D live-cell imaging to show that Oo-Mvi exhibit cellular characteristics that fit an exocrine function for signaling communication. We find that deletion of the microvilli-forming gene Radixin in oocytes leads to the loss of Oo-Mvi in ovaries, and causes a series of abnormalities in ovarian development, resulting in shortened reproductive lifespan in females. Mechanistically, we find that Oo-Mvi enrich oocyte-secreted factors and control their release, resulting in optimal selection of ovarian follicles. Taken together, our data show that the Oo-Mvi system controls the female reproductive lifespan by governing the fate of follicles.

Association of the CETP gene TaqIB and D442G polymorphisms with essential hypertension in the Chinese Mongolian population.

BACKGROUND/AIM: This study aimed to explore the associations of the cholesteryl ester transfer protein (CETP) gene TaqIB and D442G polymorphisms with essential hypertension (EH). MATERIALS AND METHODS: In this case-control study, 883 hypertensive patients and 1044 normal controls were randomly selected from the Mongolian population of China. Polymerase chain reaction (PCR) and direct sequencing of PCR products were used to identify the genotypes. Haplotype analysis was performed by estimating the haplotype frequencies using the online SHEsis package. RESULTS: The distribution frequency of the B2-G haplotype was significantly lower in the EH group than in the control group (0.7% vs. 1.9%, P = 0.001, OR = 0.359 [0.188-0.689]). Subjects with the B2B2 genotype showed significantly lower levels of total cholesterol (TC) (P < 0.05). When subgrouped by sex, male subjects with the B2B2 genotype showed significantly increased high-density lipoprotein cholesterol and decreased TC levels (P < 0.05), and those with the B2 allele showed significantly lower triglyceride levels as compared to the subjects with the B1B1 homozygote (P < 0.05). CONCLUSION: TaqIB and D442G polymorphisms of the CETP gene did not independently affect the risk of developing EH in the Chinese Mongolian population, while the B2-G haplotype obviously decreased the susceptibility to EH. The B2 allele could alter the blood lipid level and reduce the risk of developing cardiovascular diseases.

[Percutaneous absorption of aconitine and mesaconitine in extracts of Radix Aconitum kusnezoffii].

OBJECTIVE: This study was conducted to evaluate the percutaneous absorption of aconitine and mesaconitine in extracts of Radix Aconitum kusnezoffii. METHODS: The extracts of Radix Aconitum kusnezoffii were collected by using Franz diffusion cells after permeation through the skin of rats. Then rate constants of skin permeation of aconitine and mesaconitine were determined by high-performance liquid chromatography (HPLC). RESULTS: Under the condition that the concentrations of azone and propylene glycol were both 4%, the cumulative doses of skin permeation (Q) of mesaconitine and aconitine in the extracts of Radix Aconitum kusnezoffii (600 mg/ml) for 24 hours were 165.819 and 487.747 microg/cm(2) respectively, and their rate constants of skin permeation were 18.391 and 78.805 microg.cm(-2).h(-1) respectively. CONCLUSION: The aconitine and mesaconitine in the extracts of Radix Aconitum kusnezoffii can penetrate well through the skin of rats. Propylene glycol and azone can promote this penetration effects. The formula of skin permeation of mesaconitine and aconitine is in accordance with Higuchi equation and there is a linear relationship between Q and t(1/2).

Cloning, Characterization, and Expression Analysis of MyD88 in Rana dybowskii.

The myeloid differentiation factor 88 (MyD88) is the most common adaptor protein in toll-like receptor (TLR) signaling pathways and plays an important role in the innate immune system. In this report, we conducted rapid amplification of complementary DNA (cDNA) ends (RACE), multiple sequence alignment, conserved domain search, phylogenetic tree construction, and quantitative real-time PCR to obtain and analyze the full-length cDNA sequence, the amino acid sequential structures, and the expression patterns of Rana dybowskii (Rd) MyD88. The full-length cDNA of RdMyD88 is 1472 bp, with an open reading frame of 855 bp, encoding a protein of 285 amino acid residues. The RdMyD88 amino acid sequence contains a death domain (DD) and a Toll/interleukin-1 receptor (TIR) domain. RdMyD88 was calculated as a hydrophilic protein with predicted molecular mass and pI of 32.79 kDa and 6.00, respectively. Eighteen possible phosphorylation sites including eight serine residues, six tyrosine residues, and four threonine residues are predicted. Analysis of multiple sequence alignment and phylogenetic tree revealed that the predicted RdMyD88 protein is closest to its Xenopus counterparts. The PCR result showed that RdMyD88 is expressed in various tissues of R. dybowskii. Quantitative real-time PCR (qPCR) was used to examine the expression of RdMyD88 in the heart, liver, and kidney. After Rana grylio virus (RGV) exposure, the expression of RdMyD88 in the heart, liver, and kidney were significantly upregulated and reached peak levels at 48, 48, and 72 h post-infection (hpi), respectively. Meanwhile, in response to Aeromonas hydrophila (AH) infection, clear upregulation of RdMyD88 was observed in the heart, liver, and kidney and reached its peak at 48, 6, and 12 hpi, respectively. The highest levels of induction were found in the kidney after both RGV and AH infections. These findings indicate that RdMyD88 has a conserved structure and is probably an important component of the innate immunity in R. dybowskii. This report firstly characterized one adaptor molecule of the TLR signaling pathways in R. dybowskii, thereby providing reference for further researches on the amphibian innate immune system.