Different Stages Of Follicular Development Research Articles

Comparative study of the three-dimensional genomes of granulosa cells in germinal vesicle and metaphase II follicles.

Follicle development is a critical process in the female reproductive system, with significant implications for fertility and reproductive health. Germinal vesicle (GV) oocytes are primary oocytes that are arrested in the dictyate stage, also known as the diplotene stage of meiotic prophase I. Metaphase II (MII) is the stage at which the oocyte is typically retrieved for assisted reproductive technologies such as in vitro fertilization (IVF). The granulosa cells play a pivotal role in follicle development processes. 3D chromatin organization is a fundamental aspect of cellular biology that has significant implications for gene regulation and cellular function. In this study, we investigated 3D chromatin organization in granulosacells from GV and MII follicles, which is essential for understanding the regulatory mechanisms governing oocyte development. The results revealed distinct compartmentalization patterns,including stable genomic regions and transitions during oocyte maturation. Notably, there was a significant shift in functional gene activation, particularly in processes related to hormone metabolic pathways. Furthermore, alterations in topologically associating domains (TADs) were observed, with differential expression observed in genes that are involved in crucial biological processes. The analysis also identified a subset of genes with altered promoter-enhancer interactions (PEIs), reflecting a regulatory shift in gene expression related to reproductive processes. These findings provide valuable insights into 3D genome organization in granulosa cells with implications for reproductive health and the development of assisted reproductive technologies. Understanding spatial genome organization at different stages of follicular development may help realize novel strategies for enhancing success rates in assisted reproductive technologies.

In vitro growth of secondary follicles from cryopreserved-thawed ovarian cortex.

Can secondary follicles be obtained from cultured cryopreserved-thawed human ovarian cortical tissue? We obtained high-quality secondary follicles from cultured cryopreserved-thawed human ovarian cortical tissue from cis female donors (cOVA), but not from trans masculine donors (tOVA) in the same culture conditions. The in vitro growth of oocytes present in unilaminar follicles into metaphase II stage (MII) oocytes has been previously achieved starting from freshly obtained ovarian cortical tissue from adult cis female donors. This involved a multi-step culture protocol and the first step included the transition from unilaminar follicles to multilayered secondary follicles. Given that the ovarian cortex (from both cis female and trans masculine donors) used for fertility preservation is cryopreserved, it is crucial to investigate the potential of unilaminar follicles from cryopreserved-thawed ovarian cortex to grow in culture. Cryopreserved-thawed ovarian cortical tissue from adult trans masculine donors (n = 3) and adult cis female donors (n = 3) was used for in vitro culture following the first culture step described in two published culture protocols (7-8 days and 21 days) and compared to freshly isolated ovarian cortex from trans masculine donors (n = 3) and to ovarian cortex prior to culture. Ovarian cortical tissue was obtained from adult trans masculine donors undergoing gender-affirming surgery while using testosterone, and from adult cis female donors undergoing oophorectomy for fertility preservation purposes before chemotherapy. The ovarian cortex was fixed either prior (day 0) or after the culture period. Follicular survival, growth, and morphology were assessed through histology and immunofluorescence. We quantified the different stages of follicular development (primordial, primary, secondary, and atretic) after culture and observed an increase in the percentage of secondary follicles as well as an increase in COLIV deposition in the stromal compartment regardless of the culture media used. The quality of the secondary follicles obtained from cOVA was comparable to those prior to culture. However, in the same culture conditions, the secondary follicles from tOVA (fresh and cryo) showed low-quality secondary follicles, containing oocytes with small diameter, granulosa cells that expressed abnormal levels of KRT19 and steroidogenic-marker STAR and lacked ACTA2+ theca cells, when compared to tOVA secondary follicles prior to culture. The number of different donors used was limited. Our study revealed that cryopreserved-thawed cOVA can be used to generate high-quality secondary follicles after culture and those can now be further tested to evaluate their potential to generate functional MII oocytes that could be used in the clinic. However, using the same culture protocol on tOVA (fresh and cryo) did not yield high-quality secondary follicles, suggesting that either the testosterone treatment affects follicular quality or adapted culture protocols are necessary to obtain high-quality secondary follicles from tOVA. Importantly, caution must be taken when using tOVA to optimize folliculogenesis in vitro. This research was funded by the European Research Council Consolidator Grant OVOGROWTH (ERC-CoG-2016-725722 to J.S.D.V. and S.M.C.D.S.L.), the Novo Nordisk Foundation (reNEW NNF21CC0073729 to H.C., F.W., J.S.D.V., S.M.C.D.S.L.), and China Scholarship Council (CSC 202008320362 and CSC 202008450034 to H.C. and F.W.), respectively. The authors have no conflicts of interest to declare. N/A.

Treatment of ovarian damage induced by chemotherapeutic drugs in female rats with G-CSF and platelet-rich plasma(PRP): an immunohistochemical study correlation with novel marker INSL-3

Objective To assess the impacts of Platelet-Rich Plasma(PRP) and Granulocyte Colony-Stimulating Factor(G-CSF) on a rat model with induced ovarian follicular damage caused by cyclophosphamide(Cy). Materials and Methods Forty-two Sprague–Dawley rats were randomly allocated into seven distinct groups as; Group 1(control): NaCl intraperitoneal (IP) injection was administered on days D1, D7, and D14. Group 2(Cy):Cy IP injection on D1 + NaCl IP injection on D7 and D14 were administered. Group 3(PRP): PRP IP injection on D1,D7 and D14 were administered. Group 4(Cy + PRP):Cy IP injection on D1 and PRP IP injection on D1, D7 and D14 were administered. Group 5(G-CSF): G-CSF IP injection on D1, D7 and D14 were administered. Group 6(Cy + G-CSF):Cy IP injection on D1+ G-CSF IP injection on D1, D7 and D14 were administered. Group 7(Cy + PRP + G-CSF):Cy IP injection on D1+ PRP IP injection on D1,D7 and D14+ G-CSF IP injection on D1,D7 and D14 were administered. Follicular number, histological scores of AMH and INSL3 stained follicles at different stages of follicular development, and serum Anti-Müllerian hormone(AMH) were evaluated. Results The primary, secondary, and antral follicle intensity scores for AMH-positive staining were most prominent in Groups 3 and 5. There was no significant difference between groups 4, 6 and 7 compared to group 1 in terms of follicule counts and AMH staining. The intensity scores of AMH-positive staining follicles were notably reduced in group 2 compared to groups 4, 6, and 7, with a significant difference (p < .01). Among the groups, group 2 exhibited the least intense antral follicle staining for INSL3, displaying a significant difference(p < .01) compared to the remaining groups. Conclusions Autologous PRP and G-CSF might protect ovarian function in the face of ovarian damage caused by Cy-induced effects.

Evaluation of the morphology and development of preantral ovarian follicles in mice submitted to a chronic diet of dietary supplementation with Pereskia aculeata Miller leaves.

This study aimed to investigate the effect of including mouse feed with different concentrations (5, 10, or 20%) of Pereskia aculeata Miller (PAM) leaves on the morphology and development of preantral ovarian follicles and ovarian stromal cell density. The oral toxicity was performed using repeated dose toxicity assays subdivided into experiments of 30 days and 90 days of treatment. After the experiments, the ovaries of each animal were collected and submitted to classical histology. At 30 and 90 days, there was an equivalent percentage of normal, primordial, and developing follicles (P > 0.05) between PAM treatments compared to the control. Regarding the different stages of follicular development, after 90 days, there was a higher percentage (P < 0.05) of developing follicles only in the control group compared to day 30. The PAM 5% treatment was the only one that affected the cell density in the stroma after 90 days of treatment. Thus, we observed that supplementing the diet with P. aculeata did not pose any risk concerning animal consumption; specifically, there were no toxic reproductive effects observed from adding Pereskia aculeata Miller to the mouse diet.

Posthatch developmental changes in the ovary with emphasis on follicular development and atresia in the native chicken (Uttara fowl) of Uttarakhand, India.

This experiment was designed to investigate the postnatal development of the ovary in the Uttara fowl chicken and was conducted on 54 apparently healthy female birds divided into different age groups, namely Day 1 and Weeks 1, 4, 8, 12, 16, 20, 24, 28 with six birds each. During postnatal development, the left ovary gradually increased in size and complexity. The segmentation of the ovary started by 4 weeks, follicular eruption by 8 weeks, small liquor follicles (1-5 mm) appeared by 16 weeks, pre-hierarchical follicles by 20 weeks and hierarchical follicles by 24 weeks of age. The cortex was distinctly differentiated from the medulla in the early stage of ovarian development. However, the division between cortex and medulla was gradually obscured with age (transitional stage) and distinction was completely lost in the mature ovary. The different stages of follicular development in the chicken ovary were classified as primordial, primary, growing at Stage I, II and III stromal follicles besides pre-hierarchical and hierarchical surface follicles. The primordial and primary follicles showed cytoplasmic sudanophilic substances, especially in the Balbiani's yolk body, indicating the presence of lipids (Sudan Black B) with no activity for neutral polysaccharides (periodic acid Schiff method). It was observed that apoptotic changes may affect any stage of developing follicle resulting in arrested growth and atrophy. An early form of follicular atresia was the fate of the growth-arrested primordial and primary follicles, whereas the glandular form of atresia was commonly observed in growing follicles arrested at Stages I and II. The scanning electron micrographs unveiled the follicles as hollow oval structures with a follicular lumen lined by the perivitelline membrane (glycoprotein membrane) having lacunae giving a honeycomb-like appearance.

Immunolocalization of activin and inhibin at different stages of follicular development in the lizard Sceloporus torquatus

The activins and inhibins are glycoproteins with a role in the follicular development of vertebrates, that are found in follicular fluid and somatic follicular cells, with a different pattern among taxa. The principal function of activin (Act) is to modulate the follicle-stimulating hormone (FSH) synthesis and secretion, whereas inhibin (Inh) downregulates it. Both factors are modulators of intraovarian follicular recruitment, oocyte maturation, cell proliferation, and steroidogenic activity. Our aim was to characterize the immunolocalization of Act and Inh in the ovarian follicles during the reproductive cycle of the lizard Sceloporus torquatus. Act was detected in the granulosa cells and oocyte cortex in the different stages of follicular development. On the other hand, we identified Inh in the oocyte cortex and the cytoplasm of pyriform and small cells of previtellogenic follicles. Also, we found immunoreactivity in the oocyte cortex, theca, and small cells of vitellogenic and preovulatory follicles. Our data provide evidence that Act and Inh have changes related to the stage of follicular development. This dynamic appears to be conserved among vertebrates and is fundamental to ensure an adequate follicular development in this specie.

Evaluation of cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles in jennies.

Reference values of follicular fluid components at different stages of follicular development in jennies are unknown. It is important to estimate such values because the biochemical composition of follicular fluid provides a suitable microenvironment for oocyte development, growth and maturation. The experimental protocol was carried out in the Equine Reproduction Clinic of the FMVZ-UNAM and was approved by an Institutional Sub-committee for the Care and Use of Experimental Animals (SICUAE.MMVZ-2020/4-2). Seven healthy female donkeys “Mexican burro” with average body condition score of 3 on a scale of 1 to 5 (Svendsen et al. The professional Handbook of the Donkey. 2008), were monitored by transrectal ultrasonography through the Veterinary Digital Diagnostic Ultrasound System 5 MHz (Model, DUS 60 VET) with a multi-frequency linear transducer. The ovum pick up (OPU) by transvaginal aspiration (TVA) was performed in a total of 70 preovulatory follicles, 10 for each jenny using a real- time B- mode ultrasound vaginal 6.5 MHz (Sonovet 600) with curvilinear transducer and 12 gauge (12 G) needle. Follicular fluid components were measured and analyzed by Luminex Multiplex Assays at the Universidad Nacional Autónoma de México. The relationship between cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles and follicular size was assessed. The preovulatory follicles are classified in three groups accordingto the diameter: small (27-30 mm), medium (31-35 mm) and large (36-38 mm). Concentrations of cortisol were lower in the large follicles group (18.8 ng/ml) compared with small (721.44 ng/ml) and medium follicles (361 ng/ml). The concentrations of thyroxine were higher in the medium follicle group (49 ng/ml) than in the small (21ng/ml) and large follicle groups (15.9 ng/ml). Finally, the concentration of triiodothyronine was higher in the small follicles group (1043 ng/ml) followed by the medium (545 ng/ml) and the lowest concentration was in the large follicles group (22 ng/ml). It is concluded that thyroxine concentrations are lower than T4 concentrations in preovulatory follicles irrespective of size as reported for human follicular fluid (Wakim. et. al. Fertil Steril 1993; 59(6) 1187–1190). Furthermore, no relationship was found between preovulatory follicular size and thyroxine hormone concentrations. It has been suggested that cortisol and triiodothyronine concentrations are related to preovulatory follicle size. Cortisol concentrations are higher in preovulatory follicles that in subordinate follicles and cortisol is a mediator of apoptosis in granulosa cells, differentiation, development and maturation of tissues, specifically in the oocyte (Acosta & Miyamoto. Anim Reprod Sci 2004; 82:127-140, Scarlett. et al. Dom Anim Endocrinol 2016; 59:11-22). The role of cortisol within the different sizes of preovulatory follicles is unknown.

Dynamics of extracellular vesicle-coupled microRNAs in equine follicular fluid associated with follicle selection and ovulation.

Innumerable similarities in reproductive cyclicity and hormonal alterations highlight the considerable utility of the mare to study aspects of follicular dynamics and reproductive function in view of the largely constricted, human research subjects. The bi-directional communication between the growing oocyte and the surrounding somatic cells embodies the hallmark of mammalian follicular development, partially mediated by extracellular vesicles (EVs) encapsulated with microRNAs (miRNAs) and present in the follicular fluid (FF). Here, we aimed to decipher the dynamics of the miRNAs in EVs from equine FF aspirated in vivo during different stages of follicular development, namely, predeviation (PreDev; 18-20 mm), deviation (Dev; 22-25 mm), postdeviation (PostDev; 26-29 mm), preovulatory (PreOV; 30-35 mm), and impending ovulation (IMP; ∼40 mm). Approximately 176 known miRNAs were found in all groups with 144 mutually detected among all groups. Cluster analysis exhibited 15 different expression patterns during follicular development. Among these patterns, a group of 22 miRNAs (including miR-146b-5p, miR-140, and miR-143) exhibited a sharp reduction in expression from the PreDev until the PreOV stage. Another cluster of 23 miRNAs (including miR-106b, miR-199a-5p, and miR-125a-5p) exhibited a stable expression pattern at the PreDev stage until the PostDev stage, with a significant increase at the PreOV stage followed by a significant decrease at the IMP stage. In conclusion, this study provides greater insights into the stage-specific expression dynamics of FF EV-miRNAs during equine follicular development, which may propose novel approaches to improve ART and provide new biomarkers to facilitate the assessment of ovarian pathophysiological conditions.

Distribution of tetraspanins in bovine ovarian tissue and fresh/vitrified oocytes

Tetraspanin proteins are mostly known as organizers of molecular complexes on cell membranes, widely expressed on the surface of most nucleated cells. Although tetraspanins participate in many physiological processes of mammals, including reproduction, their relevance to the processes of folliculogenesis and oogenesis has not yet been fully elucidated. We bring new information regarding the distribution of tetraspanins CD9, CD81, CD151, CD82, and CD63 at different stages of follicular development in cattle. The found distribution of tetraspanin CD9, CD63, and integrin alpha V in similar areas of ovarian tissue outlined their possible cooperation. We also describe yet-unknown distribution patterns of CD151, CD82, and CD63 on immature and mature bovine oocytes. The unique localization of tetraspanins CD63 and CD82 in the zona pellucida of bovine oocytes suggested their involvement in transzonal projections. Furthermore, we present an unchanged distribution pattern of the studied tetraspanins in vitrified mature bovine oocytes. The immunofluorescent analysis was supplemented by in silico data addressing tetraspanins expression in the ovarian cells and oocytes across several species. The obtained results suggest that in the study of the oocyte development and potentially the fertilization process of cattle, the role of tetraspanins and integrins should also be taken into account.

Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors.

In this paper, we investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities on ovarian follicles of adult pigs. Piglets were injected with MXC (20 μg/kg body weight) or corn oil (controls) from postnatal Day 1 to Day 10 (n = 5 per group). Then, mRNA expression, protein abundance and immunolocalization of growth and differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), anti-Müllerian hormone (AMH) and cognate receptors (ACVR1, BMPR1A, BMPR1B, TGFBR1, BMPR2, and AMHR2), as well as FSH receptor (FSHR) were examined in preantral and small antral ovarian follicles of sexually mature gilts. The plasma AMH and FSH levels were also assessed. In preantral follicles, neonatal exposure to MXC increased GDF9, BMPR1B, TGFBR1, and BMPR2 mRNAs, while the levels of AMH and BMP15 mRNAs decreased. In addition, MXC also decreased BMP15 and BMPR1B protein abundance. Regarding small antral follicles, neonatal exposure to MXC upregulated mRNAs for BMPR1B, BMPR2, and AMHR2 and downregulated mRNAs for AMH, BMPR1A, and FSHR. MXC decreased the protein abundance of AMH, and all examined receptors in small antral follicles. GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles of control and treated ovaries. All analyzed receptors were detected in the oocytes and granulosa cells of preantral follicles, and in the granulosa and theca cells of small antral follicles. The exception, however, was FSHR, which was detected only in the granulosa cells of small antral follicles. In addition, MXC decreased the plasma AMH and FSH concentrations. In conclusion, the present study may indicate long-term effects of neonatal MXC exposure on GDF9, BMP15, AMH, and FSH signaling in ovaries of adult pigs. However, the MXC effects varied at different stages of follicular development. It seems that neonatal MXC exposure may result in accelerated initial recruitment of ovarian follicles and impaired cyclic recruitment of antral follicles.

Advances of the Regulation of microRNAs in Follicular Development

In recent years,microRNAs(miRNAs)have been detected at different stages of follicular development and in different cells of follicles.Extracellular vesicle(EV)-derived miRNAs have also been detected in the follicular fluid of mature follicles.miRNAs participate in the regulation of normal follicular development,and the regulation disorder may lead to the occurrence of some ovarian diseases.In order to further systematically elucidate the regulatory mechanism of miRNAs on follicular development and find suitable EV-derived miRNAs that can predict oocyte development,we reviewed the functions of miRNAs in follicular development from the perspectives of granulosa cell development,oocyte development,and hormone synthesis.

FSH stimulated bovine granulosa cell steroidogenesis involves both canonical and noncanonical WNT signaling

Gonadotrophins play key roles in follicular development; however, the underlying molecular mechanisms are not fully understood. Follicle stimulating hormone (FSH) regulation of aromatase and subsequent estradiol (E2) production relies on β-catenin, a key effector of WNT signaling. We previously demonstrated that treatment with the canonical WNT inhibitor, IWR-1, reduced FSH induced bovine granulosa cell E2 production in vitro. Here we demonstrated that intrafollicular injection in vivo with IWR-1 alters steroidogenesis and triggers a significant decrease in estrogen to progesterone ratio in the IWR-1 treated follicles compared to diluent injected control follicles. We next examined markers of canonical and noncanonical WNT signaling in dominant and subordinate follicles collected at different stages of follicular development and showed that protein for both CTNNB1 (canonical pathway) and phosphorylated (p)-LEF1 (noncanonical pathway) was significantly elevated in dominant compared to subordinate follicles at the early dominance stage of development. Therefore, we hypothesized that canonical and/or noncanonical WNT ligands modulate FSH stimulated E2 production. Hence, we examined the effects of specific WNT ligands on FSH stimulated E2 production in the absence of endogenous WNT production in vitro. Universal WNT signaling inhibitor, LGK-974 was able to inhibit FSH stimulation of E2 and reduce the abundance of proteins linked to canonical and noncanonical WNT pathway activation. Supplementation with the canonical ligand WNT2b did not affect the inhibitory effects of LGK-974 on FSH stimulated E2 production but rescued the LGK-974 mediated inhibition of CTNNB1 (canonical pathway) but not p-LEF1, p-JNK or p-P38 abundance (noncanonical pathway) abundance. In contrast, WNT5a treatment rescued FSH stimulated estradiol production and indices of activation of both the canonical (CTNNB1) and noncanonical (p-LEF1, p-JNK and p-P38) WNT signaling pathways in LGK-974 treated granulosa cells. Taken together, these results suggest that both canonical and noncanonical WNT pathways activation is linked to FSH stimulation of E2 production by bovine granulosa cells.

Tribbles homolog 2 regulates binding partners expression in bovine granulosa cells

The Tribbles (TRIB) family including TRIB2 are serine/threonine pseudokinases, which function as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling pathways affected by TRIB2 in granulosa cells (GC) during the ovarian follicular development is not fully defined. We previously identified and reported TRIB2 as differentially expressed in growing dominant follicles (DF) while downregulated by the luteinizing hormone (LH) surge in ovulatory follicles. In the present study, we used the yeast two-hybrid screening and in vitro co-immunoprecipitation to identify and confirm TRIB2 interactions in GC. We identified CALM1, INHBA, INPPL1, NT5E, SCD, SDHB and RAB14 as TRIB2 binding partners. RT-PCR analyses showed all TRIB2 binding partners are present in GC of DF but differently regulated throughout the different stages of follicular development. TRIB2 inhibition and overexpression using, respectively, CRISPR/Cas9 and pQE systems revealed TRIB2 differently regulates the expression of its binding partners. INPPL1 and INHBA were decreased in TRIB2-inhibited GC and increased in TRIB2-overexpressed GC (P<0.05) while NT5E and SDHB were increased following TRIB2 inhibition (P<0.05). CALM1 was increased following TRIB2 overexpression and both inhibition and overexpression of TRIB2 resulted in increased RAB14 expression (P<0.05). The regulation of these partners by TRIB2 reveal a crucial role of TRIB2 in a number of biological processes in GC, and provide a larger view of potential TRIB2-regulated signal transduction pathways and a strong evidence that TRIB2 may act as a regulator of target effectors during ovarian follicular development.

Tribbles Pseudokinase 2 (TRIB2) Regulates Expression of Binding Partners in Bovine Granulosa Cells.

Members of the Tribbles (TRIB) family of pseudokinases are critical components of intracellular signal transduction pathways in physiological and pathological processes. TRIBs, including TRIB2, have been previously shown as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling network associated with TRIB2 and its binding partners in granulosa cells during ovarian follicular development is not fully defined. We previously reported that TRIB2 is differentially expressed in growing dominant follicles while downregulated in ovulatory follicles following the luteinizing hormone (LH) surge or human chorionic gonadotropin (hCG) injection. In the present study, we used the yeast two-hybrid screening system and in vitro coimmunoprecipitation assays to identify and confirm TRIB2 interactions in granulosa cells (GCs) of dominant ovarian follicles (DFs), which yielded individual candidate binding partners including calmodulin 1 (CALM1), inhibin subunit beta A (INHBA), inositol polyphosphate phosphatase-like 1 (INPPL1), 5′-nucleotidase ecto (NT5E), stearoyl-CoA desaturase (SCD), succinate dehydrogenase complex iron sulfur subunit B (SDHB) and Ras-associated protein 14 (RAB14). Further analyses showed that all TRIB2 binding partners are expressed in GCs of dominant follicles but are differentially regulated throughout the different stages of follicular development. CRISPR/Cas9-driven inhibition along with pQE-driven overexpression of TRIB2 showed that TRIB2 differently regulates expression of binding partners, which reveals the importance of TRIB2 in the control of gene expression linked to various biological processes such as proliferation, differentiation, cell migration, apoptosis, calcium signaling and metabolism. These data provide a larger view of potential TRIB2-regulated signal transduction pathways in GCs and provide strong evidence that TRIB2 may act as a regulator of target genes during ovarian follicular development.

Co-culture model reveals the characteristics of theca cells and the effect of granulosa cells on theca cells at different stages of follicular development.

Theca cells (TCs) play an important role in follicular development, which cannot be separated from granulosa cells (GCs). However, compared with mammals, the TCs and the effects of GCs on TCs at different follicular development stages (FDSs) have specific characteristics in avian species, but none of them have been clearly defined. In this study, we established an in vitro co-culture (with GC at the corresponding stage) model of goose TCs at different FDSs (pre-hierarchical, hierarchical and F1) by using a transwell system. The properties of TCs in co-culture at the three FDSs, including cell morphology, activity and intracellular lipid content, as well as the expression of key genes involved in de novo lipogenesis, steroidogenesis, proliferation and apoptosis, were examined and defined. We further compared the mono-culture and co-culture groups. After co-culture, the activity of TCs showed significant (p<.01) increases in all stages; moreover, in pre-hierarchical TCs, the expression levels of FAS, SREBP, 3β-HSD and CCND1 were promoted, and PPARγ, CYP19, BCL2 and CAS3 were inhibited (p<.05); in the hierarchical TCs, the expression levels of PPARγ, FAS, CYP19, CCND1 and BCL2 were promoted, and SREBP, STAR, 3β-HSD and CAS3 were inhibited (p<.05), whereas in the F1 TCs, the expression levels of PPARγ, FAS, 3β-HSD, CYP19 and CCND1 were promoted, and STAR and CAS3 were inhibited (p<.05). These results suggested that GCs at the three FDSs have dynamic and complex influences on the physiological characteristics of TCs, and the influences on TCs at the three FDSs were varied.

Human Follicle in vitro Culture Including Activation, Growth, and Maturation: A Review of Research Progress.

Fertility preservation has received unprecedented attention nowadays. In addition to cryopreservation and re-implantation of embryos, oocytes, and ovarian tissue pieces, in vitro culture system for follicles/oocytes has been considered as an alternative strategy for fertility preservation. Since the metabolic dynamics and required nutrients are not entirely the same in different stages of follicular development, optimization of each culture step is needed. In this paper, literature regarding culture conditions in three steps were analyzed. Known additives in activation stage included 740Y-P, bpV(HOpic), follicle stimulating hormone (FSH), human serum albumin (HSA), ITS, growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and cyclic adenosine monophosphate (cAMP), with different degrees of activation promotion and potential detrimental effect on DNA integrity. For isolated follicles growth stage, actin A, FSH, basic fibroblast growth factor (bFGF), estradiol were proved to improve development or proliferation. As for maturation, addition of growth hormone, melatonin, C-type natriuretic peptide (CNP), GDF9, cilostamide, or forskolin helped to regulate maturation rate or improve oocyte quality. Based on previous sequential culture system for human follicles, optimization is needed to achieve higher maturation rate and better oocyte quality, pursuant to current review, which demonstrated the effects of various additives on different stages.

Human follicular fluid shows diverse metabolic profiles at different follicle developmental stages

BackgroundTo investigate the metabolic profiles in the follicular fluid (FF) samples from patients undergoing in vitro fertilization (IVF) and to analyze the correlations with follicular development.MethodsThe FF samples were obtained from participants (N = 26) who were receiving IVF under the gonadotropin-releasing hormone agonist (GnRH-a) long protocol stimulation and were collected separately from small (8–13 mm) and large (17–22 mm) follicles at the time of oocyte retrieval. Metabolomic analysis of the FF samples was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).ResultsThe results demonstrated that the size of the follicle influences the metabolic signature of the FF according to the profile and differential metabolites. Dehydroepiandrosterone (DHEA), which is enriched in steroid hormone biosynthesis, correlated negatively with the oocyte maturation rate and the high-quality embryo rate, and thus could be used to estimate the predictive diagnostic potential of follicular development.ConclusionThe FF has different metabolic characteristics in different stages of follicular development. Exploring meaningful metabolites could predict follicular development, and modifications of these metabolites could influence follicular development.

Interleukins’ expression profile changes in granulosa cells of preovulatory follicles during the postpartum period in dairy cows

The postpartum period in dairy cows is associated with a state of temporary negative energy balance and could induce functional changes into ovarian granulosa cells (GC) resulting in significant impact on the ovarian function and fertility. Yet, the regulation of interleukin receptors (ILRs) in GC as well as ILs expression profile during the postpartum period have not been fully investigated. We hypothesized that the postpartum period is associated with changes in ILs expression profile that could affect follicular development and ovulation rate. First, we aimed to investigate the expression and regulation of different IL and IL receptors in GC at different stages of follicular development and then analyse the changes in target ILs expression profile induced during the postpartum period. In the first objective, normal cycling cows were selected and GC were collected from small follicles (SF), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 h following hCG injection (OF). In the second objective, dairy cows between 50 and 70 days postpartum were randomly selected, and β-hydroxybutyrate (BHB) concentrations were measured in blood samples in order to assign cows to the BHB+ group (>1.4 mmol/L) or BHB− group (<1.2 mmol/L). GC were collected from preovulatory follicles by transvaginal aspiration. Total RNA was extracted from GC of all groups for analysis of target ILs and ILRs expression. Steady-state mRNA levels of IL4R was strongest in the DF, while IL15R expression was greatest in the OF, and IL21R showed increased steady-state mRNA levels in the corpus luteum as compared to the different groups of follicles. Overall, expression of IL1A, IL1B, IL8, IL15, IL23 and TNFα was stronger in OF as compared to DF, while IL4 and IL10 expression was stronger in SF than in DF. Similarly, expression of IL1A, IL1B, IL8, IL15, IL23, and TNFα were significantly stronger in GC of BHB+ cows than in the control, while IL4 expression was significantly reduced in BHB+ as compared to control cows. We have established an IL expression profile, which suggest a correlation with BHB levels during the postpartum period. Additionally, we have demonstrated a differential regulation of target ILRs in GC at different stages of follicular development. Overall, these data provide a better understanding of the changes that could affect follicular development and ovulation during the postpartum period and lay the ground for further investigations.

Neuropeptide Y regulates proliferation and apoptosis in granulosa cells in a follicular stage-dependent manner

BackgroundThe complex regulatory mechanism involved in ovarian follicular development is not completely understood. Neuronal neuropeptide Y (NPY) is involved in the regulation of feeding behavior, energy homeostasis, and reproduction behavior, while its function in ovarian follicular development is not clear. The objective of this study was to investigate if and how NPY regulates follicle development in the ovary.MethodsAll experiments were performed using Sprague Dawley rats. To understand NPY expression pattern at different stages of follicular development, NPY content was assessed using immunohistochemistry in individual follicles. NPY and its receptors expression pattern were evaluated in granulosa cells isolated from preantral (PA), early antral (EA) and late antral follicles (LAF). The influence of NPY on granulosa cell proliferation and apoptosis were further assessed in vitro, using Ki67- and TUNEL-positivity assays. To investigate whether NPY induced-proliferation in EA granulosa cells is mediated through the activation of NPY receptor Y5 (NPY5R) and Mitogen-activated protein kinase (MEK) signal pathway, EA granulosa cells were treated with NPY5R antagonist (CGP71683) and MEK inhibitors (PD98059 and U0126), and Ki67-positive cells were assessed.ResultsNPY protein expression was follicular stage-dependent and cell type-specific. NPY signal intensity in EA was higher than those in PA and LAF. Antral granulosa cells showed the highest signal intensity compared to mural granulosa cells, cumulus cells and theca cells. Granulosa cells NPY protein content and mRNA abundance were higher in EA than in LAF. NPY receptor contents in granulosa cells were follicular stage-dependent. While NPY reduced apoptosis of EA granulosa cells, it increased the proliferation through NPY5R and MEK pathway. In contrast, in LAF granulosa cells, NPY reduced proliferation and increased the number of apoptotic cells, with no significant effects on PA granulosa cells.ConclusionThis study is the first to evaluate the intraovarian role of NPY in granulosa cells at various stage of follicular development. These results indicate that NPY regulates granulosa cells proliferation and apoptosis in a follicular stage-dependent and autocrine manner. NPY may play a role in pathogenesis of ovarian follicular disorders.

Transcriptome analysis to unravel the gene expression profile of ovarian follicular development in Magang goose.

Magang geese exhibit a unique characteristic of follicular development, with eight largest orderly arranged pre-ovulatory follicles in the abdominal cavity. However, little is known about the mechanisms underlying this follicular development. This study aimed to compare gene expression profiles of granulosa cells (GCs) at different stages of follicular development and provide comprehensive insights into follicle selection and the mechanisms underlying the well-defined follicle hierarchy in Magang geese. GCs of large white follicles (LWFs), small yellow follicles (SYFs), F8, F4, and F1 were used for RNA-seq analysis; 374, 1117, 791, and 593 genes were differentially expressed in stages LWFs to SYFs, SYFs to F8, F8 to F4, and F4 to F1, respectively, suggesting that these genes contribute to follicle selection and development. Reliability of sequencing data was verified through qPCR analysis of 24 genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways revealed a complex mechanism that remodels the extracellular matrix and turnover of extracellular matrix components in follicular development and ovulation and involves multiple pathway, such as focal adhesion, adherens junction, and extracellular matrix–receptor interaction. Some unique characteristics were observed during the different follicular development stages. For instance, some differentially expressed genes were enriched in progesterone-mediated oocyte maturation and steroid biosynthesis from stage SYFs to F8, whereas others were enriched in actin cytoskeleton regulation and vascular smooth muscle contraction from stage F4 to F1. These findings enhance our current understanding of GC function and ovarian follicles during the key stages of follicular development.