Primordial Follicle Formation Research Articles

Single-cell transcriptomic profiling provides insights into the toxic effects of Zearalenone exposure on primordial follicle assembly.

Rationale: Zearalenone (ZEN), a pollutant in our daily diet, seriously threatens the reproductive health of humans and animals. The primordial follicle (PF) assembly in the mouse occurs during the perinatal period, which determines the whole ovarian reserve in reproductive lifespan. In the current investigation, we administered ZEN orally to perinatal mice from 16.5 days post coitum (dpc) to postnatal day 3 (PD3), and single-cell RNA sequencing (scRNA-seq) was performed on PD0 and PD3 ovarian tissues in the offspring to check ZEN toxic to primordial follicle formation at the single cell level.Methods: Ovarian tissues (in vivo) were examined by single cell RNA sequencing analysis, Immunostaining, and Western blotting. Ovarian tissues (in vitro) were examined by qRT-PCR, Immunostaining, and Western blotting.Results: We found that ZEN exposure altered the developmental trajectory of both germ cells and granulosa cells. Furthermore, after establishing the cell-cell communication network between germ cells and granulosa cells, we found that this was perturbed by ZEN exposure, especially during the Hippo signaling pathway.Conclusions: This study showed that ZEN affected the status of germ cells and granulosa cells through the Hippo signaling pathway and blocked the assembly of PFs. This research contributes to our deeper understanding of the mechanisms of toxicity in different cell types and the disruption of normal intercellular signaling by ZEN exposure.

Regulation of primordial follicle formation, dormancy, and activation in mice.

In female reproduction, the oocyte number is limited after birth. To achieve a continuous ovulatory cycle, oocytes are stored in primordial follicles. Therefore, the regulation of primordial follicle dormancy and activation is important for reproductive sustainability, and its collapse leads to premature ovarian insufficiency. In this review, we summarize primordial follicle development and the molecular mechanisms underlying primordial follicle maintenance and activation in mice. We also overview the mechanisms discovered through in vitro culture of functional oocytes, including the establishment of primordial follicle induction by environmental factors, which revealed the importance of hypoxia and compression by the extra cellular matrix (ECM) for primordial follicle maintenance in vivo.

Germ cell nests in adult ovaries and an unusually large ovarian reserve in the naked mole-rat.

The naked mole-rat (NMR, Heterocephalus glaber) is renowned for its eusociality and exceptionally long lifespan (> 30 y) relative to its small body size (35-40 g). A NMR phenomenon that has received far less attention is that females show no decline in fertility or fecundity into their third decade of life. The age of onset of reproductive decline in many mammalian species is closely associated with the number of germ cells remaining at the age of sexual maturity. We quantified ovarian reserve size in NMRs at the youngest age (6 months) when subordinate females can begin to ovulate after removal from the queen's suppression. We then compared the NMR ovarian reserve size to values for 19 other mammalian species that were previously reported. The NMR ovarian reserve at 6 months of age is exceptionally large at 108,588 ± 69,890 primordial follicles, which is more than 10-fold larger than in mammals of a comparable size. We also observed germ cell nests in ovaries from 6-month-old NMRs, which is highly unusual since breakdown of germ cell nests and the formation of primordial follicles is generally complete by early postnatal life in other mammals. Additionally, we found germ cell nests in young adult NMRs between 1.25 and 3.75 years of age, in both reproductively activated and suppressed females. The unusually large NMR ovarian reserve provides one mechanism to account for this species' protracted fertility. Whether germ cell nests in adult ovaries contribute to the NMR's long reproductive lifespan remains to be determined.

Involvement of the glucocorticoid pathways in the process of primordial follicle formation in mice

Uterine atony is a major cause of postpartum hemorrhage. We recently proposed the new histological concept of postpartum acute myometritis (PAM) for the pathophysiology of refractory uterine atony of unknown etiology, which is characterized by the diffuse activation of mast cells and the complement system as well as the massive infiltration of macrophages and neutrophils into the uterine body. We herein focused on the uterine isthmus just adjacent to the body. The isthmus becomes significantly elongated throughout pregnancy. It is composed of myocytes and fibroblasts with an extracellular matrix that forms a passive lower segment during labor. The aim of this study was to histologically examine the uterine isthmus in cases of PAM in the uterine body. Under the amniotic fluid embolism-registry program in Japan, we selected PAM cases from uterine samples obtained by cesarean hysterectomy and delivered to us for analyses between 2011 and 2017. Control tissues were collected during elective cesarean section. We investigated the isthmus tissues of these cases and performed immunohistochemistry for inflammatory cell markers, i.e. neutrophil elastase, mast cell tryptase, CD68, CD3, and C5a receptor (C5aR). The numbers of tryptase-positive degranulating mast cells, elastase-positive neutrophils, CD68-positive macrophages, and C5aR-positive cells in the isthmus were significantly higher in uteri with PAM in the body than in controls without PAM. CD3 was negative in both groups. In conclusion, inflammation and an anaphylactoid reaction were histologically detected not only in the uterine body, but in the isthmus among cases of refractory PPH of unknown etiology after cesarean section.

Ptk2b deletion improves mice folliculogenesis and fecundity via inhibiting follicle loss mediated by Erk pathway.

Ptk2b has been found playing critical roles in oocyte maturation and subsequent fertilization in vitro. But what is the exact in vivo function in reproduction still elusive. Here, by constructing Ptk2b mutant mice, we found Ptk2b was not essential for mice fertility, unexpectedly, contrary to previously reported in vitro findings, we found Ptk2b ablation significantly improved female fecundity. Follicle counting indicated that the number of primordial follicles and growing follicles in matured mice was significantly increased in the absence of Ptk2b, whereas the primordial follicle formation showed no defects. We also found this regulation was in an autophosphorylation independent pathway, as autophosphorylation site mutant mice (PTK2BY402F ) show no phenotype in female fertility. Further biochemistry studies revealed that Ptk2b ablation promotes folliculogenesis via Erk pathway mediate follicle survival. Together, we found a novel biological function of Ptk2b in folliculogenesis, which could be potentially used as a therapeutic target for corresponding infertility.

IRX3 and IRX5 collaborate during ovary development and follicle formation to establish responsive granulosa cells in the adult mouse†.

Healthy development of ovarian follicles depends on appropriate interactions and function between oocytes and their surrounding granulosa cells. Previously, we showed that double knockout of Irx3 and Irx5 (Irx3/5 DKO) in mice resulted in abnormal follicle morphology and follicle death. Further, female mouse models of individual Irx3 or Irx5 knockouts were both subfertile but with distinct defects. Notably, the expression profile of each gene suggests independent roles for each; first, they are colocalized in pre-granulosa cells during development that then progresses to include oocyte expression during germline nest breakdown and primordial follicle formation. Thereafter, their expression patterns diverge between oocytes and granulosa cells coinciding with the formulation and maturation of intimate oocyte-granulosa cell interactions. The objective of this study was to investigate the contributions of Irx5 and somatic cell-specific expression of Irx3 during ovarian development. Our results show that Irx3 and Irx5 contribute to female fertility through different mechanisms and that Irx3 expression in somatic cells is important for oocyte quality and survival. Based on evaluation of a series of genetically modified mouse models, we conclude that IRX3 and IRX5 collaborate in the same cells and then in neighboring cells to foster a healthy and responsive follicle. Long after these two factors have extinguished, their legacy enables these intercellular connections to mature and respond to extracellular signals to promote follicle maturation and ovulation.

Reduced retinoic acid synthesis accelerates prophase I and follicle activation.

In female mammals, reproductive potential is determined during fetal life by the formation of a non-renewable pool of primordial follicles. Initiation of meiosis is one of the defining features of germ cell differentiation and is well established to commence in response to retinoic acid. WIN 18,446 inhibits the conversion of retinol to retinoic acid, and therefore it was used to explore the impact of reduced retinoic acid synthesis on meiotic progression and thus germ cell development and subsequent primordial follicle formation. e13.5 mouse fetal ovaries were cultured in vitro and treated with WIN 18,446 for the first 3 days of a total of up to 12 days. Doses as low as 0.01 µM reduced transcript levels of the retinoic acid response genes Stra8 and Rarβ without affecting germ cell number. Higher doses resulted in germ cell loss, rescued with the addition of retinoic acid. WIN 18,446 significantly accelerated the progression of prophase I; this was seen as early as 48 h post treatment using meiotic chromosome spreads and was still evident after 12 days of culture using Tra98/Msy2 immunostaining. Furthermore, ovaries treated with WIN 18,446 at e13.5 but not at P0 had a higher proportion of growing follicles compared to vehicle controls, thus showing evidence of increased follicle activation. These data therefore indicate that retinoic acid is not necessary for meiotic progression but may have a role in the regulation of its progression and germ cell survival at that time and provide evidence for a link between meiotic arrest and follicle growth initiation.

Incorporation of arginine, glutamine or leucine in culture medium accelerates in vitro activation of primordial follicles in 1-day-old mouse ovary.

In vitro activation of primordial follicles provides cancer patients subjected to oncotherapy with a safe therapeutic strategy for fertility preservation, however a successful protocol for activation of primordial follicles in prepubertal patients has not yet been defined comprehensively. There is evidence that amino acids such as leucine, arginine and glutamine could stimulate the mammalian target of rapamycin (mTOR) pathway, which plays a pivotal role in primordial follicle activation. Nevertheless, there has been no report that elucidates the effect of these amino acids on in vitro development of ovarian follicles. Therefore, the present study was conducted to evaluate the effects of these amino acids and their combination on the formation and activation of primordial follicles in 1-day-old murine ovaries during an 11-day culture period. The experimental groups consisted of base medium (BM), base medium + arginine (ARG), base medium + glutamine (GLU), base medium + leucine (LEU) and base medium + a combination of arginine, glutamine and leucine (AGL). The proportions of different stages of ovarian follicles and gene expression of regulatory factors were assessed using histology and quantitative real-time PCR on days 5 and 11 of culture. The proportion of transitional and primary follicles was greater in all amino acid-treated groups compared with the BM group (P < 0.05). Moreover, leucine resulted in elevated expression of Gdf9 and Bmp15, and glutamine augmented the expression of Pi3k on day 11 of culture. In conclusion, the present study showed that inclusion of leucine, glutamine, arginine or their combination in the culture medium for murine ovarian tissue could accelerate the activation of primordial follicles and alter the expression of the corresponding factors.

Dynamics of the Transcriptome and Accessible Chromatin Landscapes During Early Goose Ovarian Development.

In contrast to the situation in mammals, very little is known about the molecular mechanisms regulating early avian ovarian development. This study aimed to investigate the dynamic changes in the histomorphology as well as the genome-wide transcriptome and chromatin accessibility landscapes of the goose ovary during late embryonic and early post-hatching stages. Results from hematoxylin-eosin, periodic acid-Schiff, and anti-CVH immunohistochemical stainings demonstrated that programmed oocyte loss, oocyte nest breakdown and primordial follicle formation, and the primordial-to-secondary follicle transition occur during the periods from embryonic day 15 (E15) to post-hatching day 0 (P0), from P0 to P4, and from P4 to P28, respectively. RNA-seq and ATAC-seq analyses revealed dynamic changes in both the ovarian transcriptome and accessible chromatin landscapes during early ovarian development, exhibiting the most extensive changes during peri-hatching oocyte loss, and moreover, differences were also identified in the genomic distribution of the differential ATAC-seq peaks between different developmental stages, suggesting that chromatin-level regulation of gene expression is facilitated by modulating the accessibility of different functional genomic regions to transcription factors. Motif analysis of developmental stage-selective peak regions identified hundreds of potential cis-regulatory elements that contain binding sites for many transcription factors, including SF1, NR5A2, ESRRβ, NF1, and THRβ, as well as members of the GATA, SMAD, and LHX families, whose expression fluctuated throughout early goose ovarian development. Integrated ATAC-seq and RNA-seq analysis suggested that the number and genomic distribution of the newly appeared and disappeared peaks differed according to developmental stage, and in combination with qRT-PCR validation potentiated the critical actions of the DEGs enriched in cell cycle, MAPK signaling, and FoxO signaling pathways during peri-hatching oocyte loss and those in ligand–receptor interaction, tissue remodeling, lipid metabolism, and Wnt signaling during primordial follicle formation and development. In conclusion, our study provides a framework for understanding the transcriptome and accessible chromatin dynamics during early avian ovarian development and a new avenue to unravel the transcriptional regulatory mechanisms that facilitate the occurrence of relevant molecular events.

FIGLA, LHX8 and SOHLH1 transcription factor networks regulate mouse oocyte growth and differentiation.

Germ-cell transcription factors control gene networks that regulate oocyte differentiation and primordial follicle formation during early, postnatal mouse oogenesis. Taking advantage of gene-edited mice lacking transcription factors expressed in female germ cells, we analyzed global gene expression profiles in perinatal ovaries from wildtype, FiglaNull, Lhx8Null and Sohlh1Null mice. Figla deficiency dysregulates expression of meiosis-related genes (e.g.Sycp3, Rad51, Ybx2) and a variety of genes (e.g.Nobox, Lhx8, Taf4b, Sohlh1, Sohlh2, Gdf9) associated with oocyte growth and differentiation. The absence of FIGLA significantly impedes meiotic progression, causes DNA damage and results in oocyte apoptosis. Moreover, we find that FIGLA and other transcriptional regulator proteins (e.g.NOBOX, LHX8, SOHLH1, SOHLH2) are co-expressed in the same subset of germ cells in perinatal ovaries and Figla ablation dramatically disrupts KIT, NOBOX, LHX8, SOHLH1 and SOHLH2 abundance. In addition, not only do FIGLA, LHX8 and SOHLH1 cross-regulate each other, they also cooperate by direct interaction with each during early oocyte development and share downstream gene targets. Thus, our findings substantiate a major role for FIGLA, LHX8 and SOHLH1 as multifunctional regulators of networks necessary for oocyte maintenance and differentiation during early folliculogenesis.

Expression and possible roles of extracellular signal-related kinases 1-2 (ERK1-2) in mouse primordial germ cell development.

In the present work, we described the expression and activity of extracellular signal-related kinases 1-2 (ERK1-2) in mouse primordial germ cells (PGCs) from 8.5–14.5 days post coitum (dpc) and investigated whether these kinases play a role in regulating the various processes of PGC development. Using immunofluorescence and immunoblotting to detect the active phosphorylated form of ERK1-2 (p-ERK1-2), we found that the kinases were present in most proliferating 8.5–10.5 dpc PGCs, low in 11.5 dpc PGCs, and progressively increasing between 12.5–14.5 dpc both in female and male PGCs. In vitro culture experiments showed that inhibiting activation of ERK1-2 with the MEK-specific inhibitor U0126 significantly reduced the growth of 8.5 dpc PGCs in culture but had little effect on 11.5–12.5 dpc PGCs. Moreover, we found that the inhibitor did not affect the adhesion of 11.5 dpc PGCs, but it significantly reduced their motility features onto a cell monolayer. Further, while the ability of female PGCs to begin meiosis was not significantly affected by U0126, their progression through meiotic prophase I was slowed down. Notably, the activity of ERK1-2 was necessary for maintaining the correct expression of oocyte-specific genes crucial for germ cells survival and the formation of primordial follicles.

Molecular analysis of the effects of steroid hormones on mouse meiotic prophase I progression

BackgroundInfertility is linked to depletion of the primordial follicle pool consisting of individual oocytes arrested at the diplotene stage of meiotic prophase I surrounded by granulosa cells. Primordial germ cells, the oocyte precursors, begin to differentiate during embryonic development. These cells migrate to the genital ridge and begin mitotic divisions, remaining connected, through incomplete cytokinesis, in clusters of synchronously dividing oogonia known as germ cell cysts. Subsequently, they enter meiosis, become oocytes and progress through prophase I to the diplotene stage. The cysts break apart, allowing individual oocytes to be surrounded by a layer of granulosa cells, forming primordial follicles each containing a diplotene arrested oocyte. A large number of oocytes are lost coincident with cyst breakdown, and may be important for quality control of primordial follicle formation. Exposure of developing ovaries to exogenous hormones can disrupt cyst breakdown and follicle formation, but it is unclear if hormones affect progression of oocytes through prophase I of meiosis.MethodsFetal ovaries were treated in organ culture with estradiol, progesterone, or both hormones, labeled for MSY2 or Synaptonemal complex protein 3 (SYCP3) using whole mount immunocytochemistry and examined by confocal microscopy. Meiotic prophase I progression was also followed using the meiotic surface spread technique.ResultsMSY2 expression in oocytes was reduced by progesterone but not estradiol or the hormone combination. However, while MSY2 expression was upregulated during development it was not a precise marker for the diplotene stage. We also followed meiotic prophase I progression using antibodies against SYCP3 using two different methods, and found that the percent of oocytes at the pachytene stage peaked at postnatal day 1. Finally, estradiol and progesterone treatment together but not either alone in organ culture increased the percent of oocytes at the pachytene stage.ConclusionsWe set out to examine the effects of hormones on prophase I progression and found that while MSY2 expression was reduced by progesterone, MSY2 was not a precise diplotene stage marker. Using antibodies against SYCP3 to identify pachytene stage oocytes we found that progesterone and estradiol together delayed progression of oocytes through prophase I.

Germ cell cysts, a fetal feature in mammals, are constitutively present in the adult armadillo.

Formation and subsequent break down of ovarian germ cell (GC) cysts is a key and an evolutionary-conserved developmental event, described in phylogenetically diverse species of invertebrates and vertebrates. In mammals, cyst break down (CBD) ends at the time of, or soon after, birth with the formation of primordial follicles enclosing single oocytes, which constitute the sole reservoir of gametes available through the whole female's reproductive life. In this study, we challenge this paradigm demonstrating the constitutive presence of a large number of cysts, enclosing two-thirty GCs, in the ovary of the adult armadillo Chaetophractus villosus, belonging to the superorder Xenarthra, one of the earliest offshoots among placentals. We also describe that (a) GCs enclosed within cysts are connected by intercellular bridges-intercellular bridges-markers of their clonal origin; (b) CBD occurs through four main phases, ending with primordial follicles containing single oocytes; (c) GCs encompass meiotic prophase I stages, from leptotene to diplotene; (d) seasonal variations in the number of GCs enclosed within cysts, suggesting the presence of a GC multiplying activity. The armadillo C. villosus''s ovary emerges as an extraordinary resource to investigate folliculogenesis and to explore the evolutionary past of the mammalian ovary.

ELAVL2-directed RNA regulatory network drives the formation of quiescent primordial follicles.

Formation of primordial follicles is a fundamental, early process in mammalian oogenesis. However, little is known about the underlying mechanisms. We herein report that the RNA-binding proteins ELAVL2 and DDX6 are indispensable for the formation of quiescent primordial follicles in mouse ovaries. We show that Elavl2 knockout females are infertile due to defective primordial follicle formation. ELAVL2 associates with mRNAs encoding components of P-bodies (cytoplasmic RNP granules involved in the decay and storage of RNA) and directs the assembly of P-body-like granules by promoting the translation of DDX6 in oocytes prior to the formation of primordial follicles. Deletion of Ddx6 disturbs the assembly of P-body-like granules and severely impairs the formation of primordial follicles, indicating the potential importance of P-body-like granules in the formation of primordial follicles. Furthermore, Ddx6-deficient oocytes are abnormally enlarged due to misregulated PI3K-AKT signaling. Our data reveal that an ELAVL2-directed post-transcriptional network is essential for the formation of quiescent primordial follicles.

Dazl determines primordial follicle formation through the translational regulation of Tex14.

Deleted in azoospermia-like (DAZL) is a germ cell RNA-binding protein that is essential for entry and progression through meiosis. The phenotype of the Dazl knockout mouse has extensive germ cell loss because of incomplete meiosis. We have created a Dazl hypomorph model using short interfering RNA knockdown in mouse fetal ovary cultures, allowing investigation of Dazl function in germ cell maturation. Dazl hypomorph ovaries had a phenotype of impaired germ cell nest breakdown with a 66% reduction in total follicle number and an increase in the proportion of primordial follicles (PMFs), with smaller oocytes within these follicles. There was no significant early germ cell loss or meiotic delay. Immunostaining of intercellular bridge component testis-expressed protein (Tex)14 showed ∼59% reduction in foci number and size, without any change in Tex14 mRNA levels. TEX14 expression was also confirmed in the human fetal ovary across gestation. Using 3'UTR-luciferase reporter assays, translational regulation of TEX14 was demonstrated to be DAZL-dependant. Dazl is therefore essential for normal intercellular bridges within germ cell nests and their timely breakdown, with a major impact on subsequent assembly of PMFs.-Rosario, R., Crichton, J. H., Stewart, H. L., Childs, A. J., Adams, I. R., Anderson, R. A. Dazl determines primordial follicle formation through the translational regulation of Tex14.

Activation of autophagy in early neonatal mice increases primordial follicle number and improves lifelong fertility†.

The number of stockpiled primordial follicles is thought to be responsible for the fate of female fertility and reproductive lifetime. We previously reported that starvation in nonsuckling early neonatal mice increases the number of primordial follicles with concomitant autophagy activation, suggesting that autophagy may accelerate the formation of primordial follicles. In this study, we attempted to upregulate the numbers of primordial follicles by administering an autophagy inducer and evaluated the progress of primordial follicle formation and their fertility during the life of the mice. To induce autophagy, mice were intraperitoneally injected with the Tat-beclin1 D-11 peptide (0.02mg/g body weight) at 6-54h or 60-84h after birth. In animals that received Tat-beclin 1 D-11 by 54h after birth, the primordial follicle numbers were significantly increased compared with the control group at 60h. The ratio of expressed LC3-II/LC3-I proteins was also significantly greater. The numbers of littermates from pregnant females that had been treated with Tat-beclin 1 D-11 were maintained at remarkably greater levels until 10months old. These results were supported by an abundance of primordial follicles at even 13-15months old.

Brangus cows have ovarian reserve parameters more like Brahman than Angus cows

Bos indicus females have more surface antral follicles than Bos taurus females; however, histological studies demonstrated no difference in total number of primordial follicles between these two biological types of cattle. Primordial follicle density in the ovary was less in Nelore ovaries compared to Angus ovaries, but no studies have examined the primordial follicle density in Bos indicus cross-bred females. It, therefore, was hypothesized that primordial follicle density in the ovary would decrease as percentage Bos indicus increased. Ovaries were collected from cross-bred Angus (n = 32, no Bos indicus influence), Brangus (n = 15), or Brahman (n = 9) cows and prepared for histological evaluation. There was no difference in total number of primordial follicles per ovary between breeds (P > 0.10). When numbers of primordial follicles were expressed on a per gram of ovarian tissue basis, there were fewer primordial follicles per gram of ovarian tissue in Brangus and Brahman cows than in Angus cows (P < 0.05). Brangus cows did not differ from Brahman cows in primordial follicle density (P > 0.10). Differences in primordial follicle density could indicate differences in capacity of ovarian stroma to produce factors necessary for oogonial proliferation and primordial follicle formation among breeds. Identifying these factors could improve the aprroach for culturing pre-antral follicles of cattle. Furthermore, these results explain why ultrasonographic antral follicle counts may need to be adjusted to a greater threshold to predict size of the ovarian reserve and determine ovarian reserve related reproductive traits in Bos indicus females.

SP1 governs primordial folliculogenesis by regulating pregranulosa cell development in mice.

Establishment of the primordial follicle (PF) pool is pivotal for the female reproductive lifespan; however, the mechanism of primordial folliculogenesis is poorly understood. Here, the transcription factor SP1 was shown to be essential for PF formation in mice. Our results showed that SP1 is present in both oocytes and somatic cells during PF formation in the ovary. Knockdown of Sp1 expression, especially in pregranulosa cells, significantly suppressed nest breakdown, oocyte apoptosis, and PF formation, suggesting that SP1 expressed by somatic cells functions in the process of primordial folliculogenesis. We further demonstrated that SP1 governs the recruitment and maintenance of Forkhead box L2-positive (FOXL2+) pregranulosa cells using an Lgr5-EGFP-IRES-CreERT2 (Lgr5-KI) reporter mouse model and a FOXL2+ cell-specific knockdown model. At the molecular level, SP1 functioned mainly through manipulation of NOTCH2 expression by binding directly to the promoter of the Notch2 gene. Finally, consistent with the critical role of granulosa cells in follicle survival in vitro, massive loss of oocytes in Sp1 knockdown ovaries was evidenced before puberty after the ovaries were transplanted under the renal capsules. Conclusively, our results reveal that SP1 controls the establishment of the ovarian reserve by regulating pregranulosa cell development in the mammalian ovary.

Gestational diabetes promotes germ cell cCyst breakdown and primordial follicle formation in newborn mice via the AKT signaling pathway.

Type 1 diabetes (T1D) is a common disease in which pancreatic β cells are impaired due to auto-immunity, pregnancy in women with it is associated with increased risk of neonatal morbidity, mortality. However, the effects of gestational diabetes on the reproduction of newborn offspring are still poorly understood. Here, we determined the cyst breakdown and primordial follicle formation in neonatal offspring born by streptozotocin (STZ)-induced diabetic or non-diabetic female mice, and found that the germ cell cyst breakdown was promoted in neonatal offspring of STZ -induced diabetic mice at postnatal Day 1, which sequentially accelerated the primordial follicle formation. Further investigation revealed that, the expression level of PI3K and p-AKT were significantly increased in ovaries of offspring born by T1D mice. These results indicated that STZ -induced gestational diabetes promotes germ cell cyst breakdown and primordial follicle formation by regulating the PI3K/AKT signaling pathway in the newborn offspring. In addition, this effect can be rescued by an insulin supplement. Taken together, our results uncover the intergenerational effects of gestational diabetes on neonatal offspring folliculogenesis, and provide an experimental model for treating gestational diabetes and its complications in neonatal offspring.

MicroRNA 92b-3p regulates primordial follicle assembly by targeting TSC1 in neonatal mouse ovaries

ABSTRACTThe primordial follicle pool, providing all oocytes available to a female throughout her reproductive life, is established perinatally. The formation of primordial follicle pool is regulated by precise transcriptional and post-transcriptional mechanisms. Recent studies have identified several microRNAs as post-transcriptional regulatory factors in the process of primordial follicle assembly. Here, we showed that miR-92b-3p was significantly upregulated in the stage of primordial follicle assembly in newborn mouse ovaries. Inhibiting miR-92b-3p suppressed the formation of primordial follicles, while overexpression of miR-92b-3p accelerated the processes of cyst breakdown and the following primordial follicle assembly. Accordingly, the expression of follicular development-related genes was reduced upon inhibiting of miR-92b-3p and increased under miR-92b-3p overexpression. Mechanistic studies identified TSC1 as a direct target of miR-92b-3p. miR-92b-3p could activate mTOR/Rps6 signaling through targeting and inhibiting TSC1 expression. In addition, knockdown of TSC1 showed an identical phenotype with that of miR-92b-3p overexpression in accelerating processes of cyst breakdown and primordial follicle formation. Thus, our work demonstrates that miR-92b-3p is a novel regulator of primordial follicle assembly by negatively regulating TSC1 in mTOR/Rps6 signaling.