Early Follicle Development Research Articles

COMPARISON OF HUMAN ANTRAL FOLLICLES OF XENOGRAFT VERSUS OVARIAN ORIGIN REVEALS DISPARATE MOLECULAR SIGNATURES

The activation, growth and maturation of oocytes to an ovulatory phase, termed folliculogenesis, is governed by the orchestrated activity of multiple specialized cells types within the ovary, yet mechanisms governing diversification and behavior of discrete cellular sub-populations within follicles are poorly understood. We set out to identify and characterize unique subsets among the granulosa (GC), theca (TC) and stromal cell fractions, and to compare the molecular profiles of these sub-populations when isolated from native ovarian versus xenograft-derived antral follicles. Small antral follicles (< 5mm) were isolated from ovaries of organ donors or patients undergoing whole ovarian resection for fertility preservation. These were compared to antral follicles that developed in long term (> 14 weeks) xenografts of cryopreserved/thawed human ovarian cortex. Immunostaining, flow cytometry, bulk and single-cell RNA sequencing were used to segregate and interrogate discrete follicle-derived cell fractions. The GC fraction was first purified from 3 small (2-3 mm) ovary-derived antral follicles and subjected to bulk RNA sequencing to identify candidate surface molecules that may specifically identify follicle cell sub-types. Candidate factors were screened using immune-staining and flow cytometry of xenograft and ovary-derived follicles. 22 unique single-cell library preparations (∼100,000 cells) representing fractions from 13 individual follicles (8 ovary origin, 5 xenograft origin) and 6 women were prepared, sequenced and analyzed. Analysis of GC, TC and stromal subpopulations deconstructed phenotypic diversification during early antral follicle development, identifying secreted factors that are differentially enriched between mural and oophorus GCs, and segregating stromal/support and steroidogenic activity between theca externa and interna, respectively. Numerous factors were differentially expressed in follicles of xenograft versus ovarian origin, highlighting the potential contribution of humoral factors to follicular development that have not been considered, to date. These data shed light on previously undescribed subpopulations of both GC and TC compartments and provide a systems level portrait of cellular diversification in early antral human follicles.

The Development of Paeonia ostii Seeds and Oil Quality Formation

Paeonia ostii is a woody oil crop with potential value as an edible oil. With the aim of acquiring systematic knowledge of the development of P. ostii seeds, the oil content, biomass, and water content of the seeds were determined. Changes in the distribution of hydrogen protons in P. ostii seeds during follicle development were traced using magnetic resonance imaging (MRI). The formation of oil bodies in the endosperm and embryo was observed using transmission electron microscopy (TEM). Dynamic changes in oleic acid, linoleic acid, and α-linolenic acid contents were assessed by gas chromatography-mass spectrometry (GC-MS). The magnetic resonance images showed that, during early follicle development [45–85 days after flowering (DAF)], a greater quantity of liquid mucus was present within the seeds, and seeds in the same follicle developed at different rates. At 95 to 115 DAF, proton density was distributed evenly in all areas of the seed. A small dark area appeared in the center of the seed, and mucus in the follicles and water in the pericarp disappeared gradually. TEM observations showed that at 45 DAF, a few oil bodies were scattered at the cell periphery in the endosperm, and oil bodies were more numerous in the embryo. With the progression of seed development, the number and size of oil bodies in the embryo and endosperm continued to increase. The fresh and dry mass of P. ostii seeds increased from 45 to 105 DAF, then decreased after 105 DAF. The moisture content decreased, whereas the oil content increased and attained 33.1% at seed maturity. The three predominant unsaturated fatty acids accumulated simultaneously and showed stages of initial accumulation (45–65 DAF) and rapid accumulation (65–105 DAF). The results suggest that 65 to 105 DAF is a critical period for unsaturated fatty acid accumulation in P. ostii seeds.

Effects of dietary supplementation with algae, sunflower oil or soybean oil on folliculogenesis in the rabbit ovary during sexual maturation

This study aimed to examine the effects of algae (rich in omega-3 fatty acids), sunflower oil (rich in omega-6 fatty acids) and soybean oil (rich in omega-6 fatty acids) on the entire folliculogenesis in juvenile and sexually mature rabbits. After weaning, rabbits were randomly divided into four experimental groups of 14 animals each. Control animals received non-supplemented pellets, while in the other groups, the pellets contained 1% marine algae, 3% sunflower oil or 3% soybean oil. Animals from each group were slaughtered at 12 weeks of age (n = 7 per group) or at 18 weeks of age (n = 7 per group). The ovaries were harvested and fixed for hematoxylin-eosin staining, immunohistochemical localization of PCNA and TUNEL assay. Algae-enriched diet markedly decreased the number of primordial and primary follicles, while addition of sunflower oil reduced the number of primary follicles in 12-week-old rabbits. The number of antral follicles was higher following algae supplementation, but lower after addition of soybean oil in that age group. Proliferating index was decreased following supplementation with algae and soybean oil in juvenile rabbits, whereas it was increased after addition of algae and decreased following vegetable oils in mature ones. Dietary PUFAs did not impact apoptosis in the rabbit ovary of both age groups. The obtained results suggest that PUFA-enriched diet regulate either early folliculogenesis or antral follicle development in rabbits that might influence reproductive performance as a consequence. It appears that observed effects are attributed to sexual maturity.

Anti-Müllerian hormone as a qualitative marker - or just quantity?

In this review, we will summarize research looking into anti-Müllerian hormone (AMH) as a measure of oocyte quality. AMH is a key factor involved in embryogenesis but also in the development of early follicles. Owing to its relatively small inter and intracycle variability, it has become a widely used method of ovarian reserve testing. In the realm of assisted reproductive technology, it has demonstrated a reliable ability to gauge the number of oocytes obtained during an in-vitro fertilization cycle. For these purposes, AMH is a readily measured quantitative tool. However, its qualitative role is as yet undefined. Although levels of this hormone have been associated with fertilization, blastulation, implantation, and clinical pregnancy rates, there is no clear link with live-birth rates. Furthermore, AMH levels do not appear to correspond with risk of fetal trisomy. AMH does show significant predictive value for the risk of premature ovarian insufficiency and time to onset of menopause.

Single-cell Transcriptome Profiling reveals Dermal and Epithelial cell fate decisions during Embryonic Hair Follicle Development.

It is estimated that 50% of men and 25% of women worldwide suffer from hair loss, and therefore it is of great significance to investigate the molecular pathways driving hair follicle de novo morphogenesis. However, due to high cellular heterogeneity and the asynchronous development of hair follicles, our current understanding of the molecular mechanisms involved in follicle development remains limited.Methods: Single-cell suspensions from the dorsal skin of E13.5 (induction stage), E16.5 (organogenesis) fetal mice, and newborn mice (cytodifferentiation stage, postnatal day 0, P0) were prepared for unbiased single-cell RNA sequencing. To delineate the single-cell transcriptional landscape during hair follicle de novo morphogenesis, we performed t-distributed Stochastic Neighbor Embedding (tSNE), pseudotime cell trajectory inference, and regulon enrichment analysis to dissect cellular heterogeneity and reveal the molecular pathways underlying major cell type cell fate decisions. To validate our analysis, we further performed immunohistochemistry analysis of the key molecules involved during hair follicle morphogenesis. Meanwhile, intercellular communication between different cell populations was inferred based on a priori knowledge of ligand-receptor pairs.Results: Based on tSNE analysis, we identified 14 cell clusters from skin tissue and delineated their cellular identity from specific gene expression profiles. By using pseudotime ordering analysis, we successfully constructed the epithelium/dermal cell lineage differentiation trajectory. For dermal cell lineage, our analysis here recapitulated the dynamic gene expression profiles during dermal condensate (DC) cell fate commitment and delineated the heterogeneity of the different dermal papilla (DP) cell populations during in utero hair follicle development. For the epithelium cell lineage, our analysis revealed the dynamic gene expression profiles of the underappreciated matrix, interfollicular epidermis (IFE), hair shaft and inner root sheath (IRS) cell populations. Furthermore, single-cell regulatory network inference and clustering analysis revealed key regulons during cell fate decisions. Finally, intercellular communication analysis demonstrated that strong intercellular communication was involved during early hair follicle development.Conclusions: Our findings here provide a molecular landscape during hair follicle epithelium/dermal cell lineage fate decisions, and recapitulate the sequential activation of core regulatory transcriptional factors (TFs) in different cell populations during hair follicle morphogenesis. More importantly, our study here represents a valuable resource for understanding the molecular pathways involved during hair follicle de novo morphogenesis, which will have implications for future hair loss treatments.

TGF-β1 sustains germ cell cyst reservoir via restraining follicle formation in the chicken.

The transforming growth factor β (TGF-β) superfamily members are important molecules that regulate many ovarian functions under normal physiological and pathological conditions. TGF-β1 and its receptors are highly expressed in the ovarian cells of many species. However, the effect of TGF-β1 on the capacity of the avian germ cell reservoir remains unknown. In this study, 5-day-old chicks were injected with TGF-β1 (2.5, 12.5, and 62.5 μg/kg body weight) for 3 days to assess the effect of TGF-β1 on early follicle development. Morphological analysis showed that treatment with TGF-β1 (12.5 μg/kg) increased the number of germ cell cysts and reduced the number of primordial and growing follicles. The diameter and area of oocytes and follicles were decreased after TGF-β1 treatment. Immunohistochemical staining of the proliferating cell nuclear antigen revealed that the ratios of the positive somatic and granulosa cells were decreased by 16.2% and 2.48%, respectively. Furthermore, more apoptotic cells were observed in the TGF-β1 group than thoseof the control by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, we cultured the 5d chicken ovaries for 3 days in vitro and found that treatment with TGF-β1 (10 ng/mL) manifested similar results as the in vivo experiment. However, the negative effect of TGF-β1 on early ovary development was rescued by treatment with a TGF-βR1 inhibitor SD208, resulting in increased expression of steroidogenic enzymes and cell cycle-regulating proteins. In conclusion, TGF-β1 could maintain the germ cell reservoir by restraining follicle activation involving reduced cell proliferation and steroidogenic enzymes gene expression at the early stage of ovarian development.

602 Sox13 is a novel marker for hair follicle development and differentiation

The Sry-related high-mobility-group box (Sox) gene encodes a transcription factor family that plays crucial roles in the determination of cell fate and organogenesis. Sox13, a group D member of the Sox family, is expressed in various tissues such as brain, cartilage, hair, kidney, pancreas, liver, kidney and blood vessels. However, knowledge of the biological roles of Sox13 is largely limited. To examine the precise expression domains of Sox13 protein and its role in mouse development, we generated Sox13-LacZ-knock-in mice (Sox13LacZ/+), in which the Sox13 coding sequence was disrupted by an in-frame insertion of the b-galactosidase (LacZ) gene. Using the knock-in mice, we examined spatiotemporal expression patterns of Sox13 in the hair follicle and its role in hair development. X-gal staining in Sox13LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode at E14.5, and subsequently in the hair germ and the hair peg during hair follicle morphogenesis. Immunohistochemistry with an anti-b-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that during the postnatal phases of hair development Sox13 was predominantly expressed in the outer root sheath. However, the integumentary structures of Sox13LacZ/LacZ mice were grossly and histologically normal. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a novel marker for early hair follicle development and differentiation.

Sox13 is a novel early marker for hair follicle development

Sox13, a group D member of the Sry-related high-mobility group box (Sox) transcription factor family, is expressed in various tissues including the hair follicle. However, its spatiotemporal expression patterns in the hair follicle and its role in hair development remain to be elucidated. To address these questions, we generated Sox13-LacZ-knock-in mice (Sox13LacZ/+), in which the LacZ reporter gene was inserted in-frame into exon 2, which contains the translation initiation codon. X-gal staining in Sox13LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode, and subsequently in the hair germ and the hair peg during early hair follicle development. In postnatal catagen and anagen, Sox13 was detected in the epithelial sheath, whereas in telogen, Sox13 was localized in the bulge region, where hair follicle stem cells reside. Immunohistochemistry with an anti-β-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that Sox13 was predominantly expressed in the outer root sheath in anagen. However, the integumentary structures of Sox13LacZ/LacZ mice were grossly and histologically indistinguishable from those of wild type mice. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a useful marker for early hair follicle development.

Adipose-derived stem cell-secreted factors promote early stage follicle development in a biomimetic matrix.

Development of primary follicles in vitro benefits from a three-dimensional matrix that is enriched with paracrine factors secreted from feeder cells and mimics the in vivo environment. In this study, we investigated the role of paracrine signaling from adipose-derived stem cells (ADSCs) in supporting primary follicle development in a biomimetic poly(ethylene glycol) (PEG)-based matrix. Follicles co-cultured with ADSCs and follicles cultured in conditioned medium from ADSCs encapsulated in gels (3D CM) exhibited significantly (p < 0.01 and p = 0.09, respectively) improved survival compared to follicles cultured in conditioned medium collected from ADSCs cultured in flasks (2D CM) and follicles cultured without paracrine support. The gene expression of ADSCs suggested that the stem cells maintained their multipotency in the 3D PEG environment over the culture period, regardless of the presence of the follicles, while under 2D conditions the multipotency markers were downregulated. The differences in cytokine signatures of follicles exposed to 3D and 2D ADSC paracrine factors suggest that early cytokine interactions are key for follicle survival. Taken together, the biomimetic PEG scaffold provides a three-dimensional, in vivo-like environment to induce ADSCs to secrete factors which promote early stage ovarian follicle development and survival.

Contribution of the immune system to follicle differentiation, ovulation and early corpus luteum formation.

Much of what we know about the involvement of the immune system in periovulatory follicle differentiation, ovulation and subsequent formation of the corpus luteum in cattle is drawn from the findings of studies in several mammalian livestock species. By integrating published histological data from cattle, sheep and pigs and referring back to the more comprehensive knowledge bank that exists for mouse and humans we can sketch out the key cells of the immune system and the cytokines and growth factors that they produce that are involved in follicle differentiation and luteinization, ovulation and early follicle development. These contributions are reviewed and the key findings, discussed.

Dynamic expression patterns of Irx3 and Irx5 during germline nest breakdown and primordial follicle formation promote follicle survival in mouse ovaries.

Women and other mammalian females are born with a finite supply of oocytes that determine their reproductive lifespan. During fetal development, individual oocytes are enclosed by a protective layer of granulosa cells to form primordial follicles that will grow, mature, and eventually release the oocyte for potential fertilization. Despite the knowledge that follicles are dysfunctional and will die without granulosa cell-oocyte interactions, the mechanisms by which these cells establish communication is unknown. We previously identified that two members of the Iroquois homeobox transcription factor gene family, Irx3 and Irx5, are expressed within developing ovaries but not testes. Deletion of both factors (Irx3-Irx5EGFP/Irx3-Irx5EGFP) disrupted granulosa cell-oocyte contact during early follicle development leading to oocyte death. Thus, we hypothesized that Irx3 and Irx5 are required to develop cell-cell communication networks to maintain follicle integrity and female fertility. A series of Irx3 and Irx5 mutant mouse models were generated to assess roles for each factor. While both Irx3 and Irx5 single mutant females were subfertile, their breeding outcomes and ovary histology indicated distinct causes. Careful analysis of Irx3- and Irx5-reporter mice linked the cause of this disparity to dynamic spatio-temporal changes in their expression patterns. Both factors marked the progenitor pre-granulosa cell population in fetal ovaries. At the critical phase of germline nest breakdown and primordial follicle formation however, Irx3 and Irx5 transitioned to oocyte- and granulosa cell-specific expression respectively. Further investigation into the cause of follicle death in Irx3-Irx5EGFP/Irx3-Irx5EGFP ovaries uncovered specific defects in both granulosa cells and oocytes. Granulosa cell defects included poor contributions to basement membrane deposition and mis-localization of gap junction proteins. Granulosa cells and oocytes both presented fewer cell projections resulting in compromised cell-cell communication. Altogether, we conclude that Irx3 and Irx5 first work together to define the pregranulosa cell population of germline nests. During primordial follicle formation, they transition to oocyte- and granulosa cell-specific expression patterns where they cooperate in neighboring cells to build the foundation for follicle integrity. This foundation is left as their legacy of the essential oocyte-granulosa cell communication network that ensures and ultimately optimizes the integrity of the ovarian reserve and therefore, the female reproductive lifespan.

FOXL2C134W-Induced CYP19 Expression via Cooperation With SMAD3 in HGrC1 Cells.

Germline knockout studies in female mice demonstrated an essential role for forkhead box L2 (FOXL2) in early follicle development, whereas an inducible granulosa cell (GC)-specific deletion of Foxl2 in adults has shown ovary-to-testis somatic sex reprogramming. In women, over 120 different germline mutations in the FOXL2 gene have been shown to cause blepharophimosis/ptosis/epicantus inversus syndrome associated with or without primary ovarian insufficiency. By contrast, a single somatic mutation (FOXL2C134W) accounts for almost all adult-type GC tumors (aGCTs). To test the hypothesis that FOXL2C134W differentially regulates the expression of aGCT markers, we investigated the effect of FOXL2C134W on inhibin B and P450 aromatase expression using a recently established human GC line (HGrC1), which we now show to bear two normal alleles of FOXL2. Neither FOXL2wt nor FOXL2C134W regulate INHBB messenger RNA (mRNA) expression. However, FOXL2C134W selectively displays a 50-fold induction of CYP19 mRNA expression dependent upon activin A. Mechanistically, the CYP19 promoter is activated in a similar way by FOXL2C134W interaction with SMAD3, but not by FOXL2wt. SMAD2 had no effect. Moreover, FOXL2C134W interactions with SMAD3 and with the FOX binding element located at -199 bp upstream of the ATG initiation codon of CYP19 are more sustainable than FOXL2wt. Thus, FOXL2C134W potentiates CYP19 expression in HGrC1 cells via enhanced recruitment of SMAD3 to a proximal FOX binding element. These findings may explain the pathophysiology of estrogen excess in patients with aGCT.

Anethole reduces oxidative stress and improves in vitro survival and activation of primordial follicles.

Primordial follicles, the main source of oocytes in the ovary, are essential for the maintenance of fertility throughout the reproductive lifespan. To the best of our knowledge, there are no reports describing the effect of anethole on this important ovarian follicle population. The aim of the study was to investigate the effect of different anethole concentrations on the in vitro culture of caprine preantral follicles enclosed in ovarian tissue. Randomized ovarian fragments were fixed immediately (non-cultured treatment) or distributed into five treatments: α-MEM+ (cultured control), α-MEM+ supplemented with ascorbic acid at 50 μg/mL (AA), and anethole at 30 (AN30), 300 (AN300), or 2000 µg/mL (AN2000), for 1 or 7 days. After 7 days of culture, a significantly higher percentage of morphologically normal follicles was observed when anethole at 2000 μg/mL was used. For both culture times, a greater percentage of growing follicles was observed with the AN30 treatment compared to AA and AN2000 treatments. Anethole at 30 and 2000 µg/mL concentrations at days 1 and 7 of culture resulted in significantly larger follicular diameter than in the cultured control treatment. Anethole at 30 µg/mL concentration at day 7 showed significantly greater oocyte diameter than the other treatments, except when compared to the AN2000 treatment. At day 7 of culture, levels of reactive oxygen species (ROS) were significantly lower in the AN30 treatment than the other treatments. In conclusion, supplementation of culture medium with anethole improves survival and early follicle development at different concentrations in the caprine species.

Role of activin C in normal ovaries and granulosa cell tumours of mice and humans.

Activins and inhibins play important roles in the development, growth and function of the ovary. Mice lacking inhibin develop granulosa cell tumours in their ovaries that secrete activin A, and these tumours are modulated by increased activin C expression. The aim of the present study was to identify where activin C is expressed in mouse and human ovaries and whether overexpression of activin C modulates normal follicular development in mice. Immunohistochemical staining for the activin βC subunit was performed on sections from mouse and human ovaries and human adult granulosa cell tumours. Stereology techniques were used to quantify oocyte and follicular diameters, and the percentage of different follicular types in ovaries from wild-type mice and those underexpressing inhibin α and/or overexpressing activin C. Staining for activin βC was observed in the oocytes, granulosa cells, thecal cells and surface epithelium of mouse and human ovaries, and in the granulosa-like cells of adult granulosa cell tumours. Overexpression of activin C in mice did not alter follicular development compared with wild-type mice, but it did modulate the development of abnormal early stage follicles in inhibin α-null mice. These results provide further evidence of a role for activin C in the ovary.

Activated ovarian endothelial cells promote early follicular development and survival

BackgroundNew data suggests that endothelial cells (ECs) elaborate essential “angiocrine factors”. The aim of this study is to investigate the role of activated ovarian endothelial cells in early in-vitro follicular development.MethodsMouse ovarian ECs were isolated using magnetic cell sorting or by FACS and cultured in serum free media. After a constitutive activation of the Akt pathway was initiated, early follicles (50–150 um) were mechanically isolated from 8-day-old mice and co-cultured with these activated ovarian endothelial cells (AOEC) (n = 32), gel (n = 24) or within matrigel (n = 27) in serum free media for 14 days. Follicular growth, survival and function were assessed.ResultsAfter 6 passages, flow cytometry showed 93% of cells grown in serum-free culture were VE-cadherin positive, CD-31 positive and CD 45 negative, matching the known EC profile. Beginning on day 4 of culture, we observed significantly higher follicular and oocyte growth rates in follicles co-cultured with AOECs compared with follicles on gel or matrigel. After 14 days of culture, 73% of primary follicles and 83% of secondary follicles co-cultured with AOEC survived, whereas the majority of follicles cultured on gel or matrigel underwent atresia.ConclusionsThis is the first report of successful isolation and culture of ovarian ECs. We suggest that co-culture with activated ovarian ECs promotes early follicular development and survival. This model is a novel platform for the in vitro maturation of early follicles and for the future exploration of endothelial-follicular communication.CapsuleIn vitro development of early follicles necessitates a complex interplay of growth factors and signals required for development. Endothelial cells (ECs) may elaborate essential “angiocrine factors” involved in organ regeneration. We demonstrate that co-culture with ovarian ECs enables culture of primary and early secondary mouse ovarian follicles.

Ovarian transport temperature (4 vs 33 °C) impacts differently the in vitro development of isolated goat preantral and antral follicles

The aim of the present study was to determine the effect of transport temperature of goat ovaries on the in vitro survival and development of preantral follicles and early antral follicles. From each ovarian pair, the ovaries were individually transported in HEPES-buffered MEM at two different temperatures (4 vs 33 °C). The follicles were isolated at the same temperature that the ovary was transported and subsequently cultured in vitro for 18days. After follicle culture, the cumulus-oocyte complexes were recovered and submitted to in vitro maturation for 32h followed by the evaluation of oocyte viability and chromatin configuration. Our results showed that there was no effect of transport temperature (4 vs 33°C) on the percentage of morphologically normal follicles regardless of the evaluated follicular category. Preantral follicles from ovaries transported at 4°C showed a delay in the antrum formation at day 12 (78.33%) in relation to 33°C (91.94%). The overall growth rates for the two tested follicular categories (Preantral follicles and Early antral follicles) transported at 33°C was higher than those transported at 4°C. Zona pellucida thickness values were lower (P<0.05) in the oocytes from early antral follicles transported at 4°C compared to oocytes harvested from preantral follicles and early antral follicles transported at 33°C. Oocyte diameter of early antral follicles transported at 4°C was significantly higher after culture. Meiotic resumption rate was higher when preantral follicles were obtained from ovaries transported at 4°C than transported at 33°C. Finally, it was observed a significant higher ROS production only for early antral follicles at day 6 when the ovaries were previously transported at 4°C than transported at 33°C. In conclusion, the transport temperature differentially impacts the in vitro development of goat preantral follicles and early antral follicles. Even though high transport temperature (33°C) improves antrum formation and follicular growth irrespective of follicular category (Preantral follicles and Early antral follicles); the best outcome for fully grown oocyte production and meiotic resumption were obtained using low (4°C) and high (33°C) temperature for preantral follicles and early antral follicles, respectively.

Stromal Senp1 promotes mouse early folliculogenesis by regulating BMP4 expression

BackgroundMammalian folliculogenesis, maturation of the ovarian follicles, require both growth factors derived from oocyte and surrounding cells, including stromal cells. However, the mechanism by which stromal cells and derived factors regulate oocyte development remains unclear.ResultsWe observed that SENP1, a small ubiquitin-related modifier (SUMO)-specific isopeptidase, was expressed in sm22α-positive stromal cells of mouse ovary. The sm22α-positive stromal cells tightly associated with follicle maturation. By using the sm22α-specific Cre system, we show that mice with a stromal cell-specific deletion of SENP1 exhibit attenuated stroma-follicle association, delayed oocyte growth and follicle maturation with reduced follicle number and size at early oocyte development, leading to premature ovarian failure at late stages of ovulating life. Mechanistic studies suggest that stromal SENP1 deficiency induces down-regulation of BMP4 in stromal cells concomitant with decreased expression of BMP4 receptor BMPR1b and BMPR2 on oocytes.ConclusionsOur data support that protein SUMOylation-regulating enzyme SENP1 plays a critical role in early ovarian follicle development by regulating gene expression of BMP4 in stroma and stroma-oocyte communication.

Hierarchical patterning modes orchestrate hair follicle morphogenesis.

Two theories address the origin of repeating patterns, such as hair follicles, limb digits, and intestinal villi, during development. The Turing reaction–diffusion system posits that interacting diffusible signals produced by static cells first define a prepattern that then induces cell rearrangements to produce an anatomical structure. The second theory, that of mesenchymal self-organisation, proposes that mobile cells can form periodic patterns of cell aggregates directly, without reference to any prepattern. Early hair follicle development is characterised by the rapid appearance of periodic arrangements of altered gene expression in the epidermis and prominent clustering of the adjacent dermal mesenchymal cells. We assess the contributions and interplay between reaction–diffusion and mesenchymal self-organisation processes in hair follicle patterning, identifying a network of fibroblast growth factor (FGF), wingless-related integration site (WNT), and bone morphogenetic protein (BMP) signalling interactions capable of spontaneously producing a periodic pattern. Using time-lapse imaging, we find that mesenchymal cell condensation at hair follicles is locally directed by an epidermal prepattern. However, imposing this prepattern’s condition of high FGF and low BMP activity across the entire skin reveals a latent dermal capacity to undergo spatially patterned self-organisation in the absence of epithelial direction. This mesenchymal self-organisation relies on restricted transforming growth factor (TGF) β signalling, which serves to drive chemotactic mesenchymal patterning when reaction–diffusion patterning is suppressed, but, in normal conditions, facilitates cell movement to locally prepatterned sources of FGF. This work illustrates a hierarchy of periodic patterning modes operating in organogenesis.

Transcriptome response of previtellogenic ovary in Anguilla japonica after artificial hormone injection

In this study we investigate the intra-ovarian pathways underlying early follicle development in Japanese eels, Anguilla japonica. We conducted high-throughput transcriptome analyses in the initial development of the ovary via the next-generation sequencing (NGS). Japanese eels were treated with three weekly salmon pituitary homogenate (SPH) injections. Using RNA-seq, we obtained 29,117,237 and 41,867,557 reads from the control and the SPH-injected groups, respectively. Combining these RNA-seq datasets, we acquired a total of 101,711 unigenes (N50=1,517bp) after performing de novo assembly. After differentially expressed gene (DEG) analysis, 4,211 and 7,059 annotated genes showed upregulation and downregulation respectively in SPH-injected ovarian tissues. Furthermore, functions of annotated genes were classified by GO and KEGG analyses. The PTEN/PI3K-Akt pathway, Tsc/mTOR signaling, oocyte meiosis and reproduction functions were found in data of differentially expressed genes.

Follicular dynamics, ovarian vascularity and luteal development in mares with early or late postpartum ovulation

Follicular development and deviation processes during the postpartum period are not completely known in horses. Thus, we aimed to study the characteristics of follicular dynamics and ovarian vascular perfusion during the postpartum period in mares that demonstrated estrous behavior and had early (<10 days) or late (≥10 days) postpartum ovulation. Ten mares were scanned daily by transrectal ultrasonography from the first day postpartum (d1) to the sixteenth day after the first postpartum ovulation (D0 = ovulation). The animals were split in the early (n = 3) and late (n = 7) ovulation groups (average interval between parturition and ovulation: 8.0 ± 0.0 and 14.7 ± 1.2 days, respectively). For the follicular growth, no difference (P > 0.1) was detected between the groups when the data were normalized for the days preceding the first postpartum ovulation (from D−7 to D−1). However, when the data were normalized to days postpartum, the dominant follicle was larger (P < 0.05) in the early-ovulated group in all days during this period (d1 to d7). The number of follicles >25 mm diameter was greater (P < 0.05) in the early-ovulated group during the first 3 days postpartum, and the late-ovulated mares showed greater number of follicles with 20–25 mm during d4 to d7. For blood flow characteristics, no difference (P > 0.1) was detected between groups in vascular perfusion of the dominant follicle or in the ovarian pedicle ipsilateral to the largest follicle. Similarly, no difference (P > 0.1) was detected in progesterone concentrations, corpus luteum (CL) area and vascular perfusion of the CL. Pregnancy rate did not differ (P > 0.1) between the early (3/3; 100%) and late (5/7; 71.4%) groups. Therefore, the characteristics of the follicle growth on the preceding days of ovulation were similar between the early- and late-ovulated mares and were consistent with the follicular dynamics expected in non-pregnant and non-lactating mares. However, when the data were analyzed for the days relative to parturition, greater follicle development was present in mares that ovulate earlier during the postpartum period (<10 days). The results suggest that important events may occur before parturition, resulting in early follicle development, mainly in those mares that show estrus and ovulate within 10 days postpartum.