Changes in the transcriptome of bovine ovarian cortex during follicle activation in vitro

The signals that regulate activation, a key transition in ovarian follicular development, are still not well understood, especially in nonrodent species. To gain insight into the regulation of this transition in cattle, we combined a microarray approach with an in vitro system in which ovarian cortical pieces cultured in control medium are enriched for primordial follicles, whereas pieces cultured with insulin are enriched for primary follicles. Total RNA was extracted from cultured cortical pieces, and then transcripts were identified and analyzed using the Affymetrix Bovine Genome GeneChip array. Around 65% of the transcripts in the bovine GeneChip were detected in cultured cortical pieces. Comparison between pieces cultured with or without insulin generated 158 differentially expressed transcripts. Compared with controls, 90 transcripts were upregulated and 68 were downregulated by insulin. These transcripts are involved in many biological processes and functions, but most are associated with cellular growth or cell cycle/cell death. The transcript encoding ubiquitin-conjugating enzyme E2C (UBE2C) was significantly upregulated during follicle activation, and Ingenuity Pathways Analysis revealed that UBE2C can interact with the tumor suppressor phosphatase and tensin homolog (PTEN). Both PTEN mRNA and protein were lower in cortical pieces cultured with insulin than in controls. In addition, FOXO3a, a downstream effector of PTEN signaling, underwent nuclear-cytoplasmic shuttling during primordial to primary follicle development in bovine fetal ovaries, further suggesting the involvement of the PTEN pathway in follicle activation in cattle. Genes and pathways identified in this study provide interesting candidates for further investigation of mechanisms underlying follicle activation.

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Supplementation of granulosa cells conditioned medium with pyruvate and testosterone could improve early follicular development in cultured 1-day-old mouse ovaries

Zygote ◽

10.1017/s0967199421000174 ◽

2021 ◽

pp. 1-8

Author(s):

Mohammad Jafari Atrabi ◽

Parimah Alborzi ◽

Vahid Akbarinejad ◽

Rouhollah Fathi

Keyword(s):

Conditioned Medium ◽

Ovarian Tissue ◽

Follicular Development ◽

Primordial Follicle ◽

Ovarian Tissue Cryopreservation ◽

Primordial Follicles ◽

Patients With Cancer ◽

Primordial Follicle Activation ◽

Follicle Activation

Summary In vitro activation of primordial follicles could serve as a safe method to preserve fertility in patients with cancer subjected to ovarian tissue cryopreservation during oncotherapy, however the culture medium for this purpose requires to be optimized. Granulosa cell conditioned medium (GCCM) has been recognized to enhance primordial follicle activation and the present study was conducted to understand whether addition of pyruvate, a combination of insulin, transferrin and selenium (ITS) or testosterone to GCCM could improve its efficiency in this regard. To this end, 1-day-old mouse ovaries were cultured in four different media including CON (control; containing GGCM only), PYR (containing GCCM plus pyruvate), ITS (containing GCCM plus ITS) or TES (containing GCCM plus testosterone) for 11 days. Furthermore, follicular dynamics and gene expression of factors involved in follicular development were assessed using histological examination and RT-PCR, respectively, on days 5 and 11 of culture. Pyruvate decreased follicular activation, but it enhanced the progression of follicles to the primary stage. Moreover, it upregulated Bmp15 and Cx37 (P < 0.05). In the ITS group, activation of follicles was not affected and total number of follicles was reduced by day 11 of culture. Additionally, ITS downregulated Pi3k, Gdf9, Bmp15 and Cx37 (P < 0.05). Although testosterone did not affect primordial follicle activation, it enhanced the development of follicles up to the preantral stage (P < 0.05). Furthermore, testosterone inhibited the expression of Pten but stimulated the expression of Gdf9 and Cx37 (P < 0.05). In conclusion, the present study revealed that inclusion of pyruvate and testosterone into GCCM could enhance the early development of follicles in cultured 1-day-old mouse ovaries.

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Single-Oocyte Gene Expression Suggests That Curcumin Can Protect the Ovarian Reserve by Regulating the PTEN-AKT-FOXO3a Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22126570 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6570

Author(s):

Yue Lv ◽

Rui-Can Cao ◽

Hong-Bin Liu ◽

Xian-Wei Su ◽

Gang Lu ◽

...

Keyword(s):

Gene Expression ◽

Ovarian Reserve ◽

Primordial Follicle ◽

Term Treatment ◽

New Drugs ◽

Gene Profiling ◽

Primordial Follicles ◽

Long Term Treatment ◽

Follicle Activation

A better understanding of the mechanism of primordial follicle activation will help us better understand the causes of premature ovarian insufficiency (POI), and will help us identify new drugs that can be applied to the clinical treatment of infertility. In this study, single oocytes were isolated from primordial and primary follicles, and were used for gene profiling with TaqMan array cards. Bioinformatics analysis was performed on the gene expression data, and Ingenuity Pathway Analysis was used to analyze and predict drugs that affect follicle activation. An ovarian in vitro culture system was used to verify the function of the drug candidates, and we found that curcumin maintains the ovarian reserve. Long-term treatment with 100 mg/kg curcumin improved the ovarian reserve indicators of AMH, FSH, and estradiol in aging mice. Mechanistic studies show that curcumin can affect the translocation of FOXO3, thereby inhibiting the PTEN-AKT-FOXO3a pathway and protecting primordial follicles from overactivation. These results suggest that curcumin is a potential drug for the treatment of POI patients and for fertility preservation.

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In-vitro fragmentation of ovarian tissue activates primordial follicles through the Hippo pathway

Human Reproduction Open ◽

10.1093/hropen/hoaa048 ◽

2020 ◽

Vol 2020 (4) ◽

Author(s):

C De Roo ◽

S Lierman ◽

K Tilleman ◽

P De Sutter

Keyword(s):

Tissue Culture ◽

Ovarian Tissue ◽

Hippo Pathway ◽

Primordial Follicle ◽

Akt Pathway ◽

Primordial Follicles ◽

Primordial Follicle Activation ◽

The Hippo Pathway ◽

Follicle Activation

Abstract STUDY QUESTION What is the role of the Hippo and PI3K/Akt pathway in follicles during ovarian tissue culture in tissue derived from oncological patients and transgender men? SUMMARY ANSWER Results highlight a Hippo pathway driven primordial follicle activation in vitro, predominantly from Day 0 to Day 4. WHAT IS KNOWN ALREADY In-vitro ovarian tissue culture aims at activating and maturing primordial follicles for fertility restoration in patients with a threatened ovarian reserve. Not all patients are eligible for ovarian cortex transplantation and therefore several groups are attempting to culture ovarian tissue in-vitro. Cortex fragmentation disrupts the Hippo pathway, leading to increased expression of downstream growth factors and follicle growth. The PI3K/Akt pathway is considered the intracellular pathway to where different extracellular factors involved in primordial follicle activation in-vivo converge. In order to optimise current ovarian tissue culture models, information on progression of these pathways during tissue culture is mandatory. STUDY DESIGN, SIZE, DURATION The first step of a multistep cortex culture system was performed using 144 ovarian cortex pieces from a total of six patients. Per patient, 24 cortical strips were cultured for 6 days and six pieces per patient were collected for downstream analysis of follicle development and Hippo and PI3K/Akt pathway targets every second day. PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian tissue was obtained from oncological (N = 3; 28.67 ± 4.51 years) and transgender (N = 3; 23.33 ± 1.53 years) patients. Follicles were analysed using haematoxylin-eosin staining and pathways were studied using immunohistochemistry and precise follicle excision by laser capture micro-dissection for RT-qPCR analysis. MIQE guidelines for RT-qPCR were pursued. Reference gene selection (GAPDH, RPL3A, 18s rRNA) was performed using GeNorm Reference Gene Selection Kit. Statistical analysis was conducted with IBM SPSS Statistics 23 (Poisson regression, negative binomial regression, ANOVA and paired t-test). MAIN RESULTS AND THE ROLE OF CHANCE Immunohistochemical analysis confirmed a Hippo pathway driven primordial follicle activation due to mechanical manipulation of the cortical strips. Ovarian tissue preparation and culture induced the inhibitory phosphorylated Yes-associated protein (pYAP) to disappear in granulosa cells of primordial follicles on Day 2. The stimulatory YAP on the contrary appeared in primordial granulosa cells over increasing culture days. Looking at the YAP target connective tissue growth factor (CTGF), a significantly up-regulated CTGF was noted in primordial follicles when comparing Day 2 and Day 4 (ratio Day 2/4 = 0.082; P < 0.05), clearly showing an effect on the Hippo pathway in primordial follicles during tissue culture. Follicle classification showed a significant drop in estimated primordial follicle counts in the oncological cohort (−78%; P = 0.021) on Day 2 and in the transgender cohort on Day 4 (−634%; P = 0.008). Intermediate follicle counts showed a non-significant increasing trend to during culture and this follicle recruitment and growth resulted in a significant rise in estimated primary follicle counts on Day 6 in oncological patients (170%; P = 0.025) and, although limited in absolute numbers, a significant increase in secondary follicles on Day 4 (367%; P = 0.021) in the transgender cohort. Subsequent antral follicle development could not be observed. LIMITATIONS, REASONS FOR CAUTION A limitation is the small sample size, inherent to this study subject, especially as a large amount of tissue was needed per patient to reduce inter-patient variation in different downstream analysis techniques. A particular and specific weakness of this study is the inability to include an age-matched control group. WIDER IMPLICATIONS OF THE FINDINGS These findings support an adapted tissue preparation for Hippo pathway disruption and a shorter first phase of tissue culture. This work may also have implications for transplantation of cryopreserved tissue as larger strips (and thus slower burnout due to less Hippo pathway disruption) could be a benefit. STUDY FUNDING/COMPETING INTEREST(S) This research was financially supported by the Foundation Against Cancer (Stichting tegen Kanker, TBMT001816N), the Flemish Foundation of Scientific Research (FWO Vlaanderen, FWO G0.065.11N10) and the Gender Identity Research and Education Society (GIRES) foundation. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

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Pentachloronitrobenzene alters progesterone production and primordial follicle recruitment in cultured granulosa cells and rat ovary†

Biology of Reproduction ◽

10.1093/biolre/ioz195 ◽

2019 ◽

Vol 102 (2) ◽

pp. 511-520

Author(s):

Yanrong Kuai ◽

Xiaobo Gao ◽

Huixia Yang ◽

Haiyan Luo ◽

Yang Xu ◽

...

Keyword(s):

Protein Kinase ◽

Granulosa Cells ◽

Crop Production ◽

Follicular Development ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Serum Progesterone ◽

Primordial Follicles ◽

Progesterone Production

Abstract Pentachloronitrobenzene (PCNB) is an organochlorine fungicide widely used for crop production and has become an environmental concern. Little is known about the effect of PCNB on ovarian steroidogenesis and follicular development. We found that PCNB stimulated Star expression and progesterone production in cultured rat granulosa cells in a dose-dependent manner. PCNB activated mitogen-activated protein kinase (MAPK3/1) extracellulat regulated kinase (ERK1/2), thus inhibition of either protein kinase A (PKA) or MAPK3/1 signaling pathway significantly attenuated progesterone biosynthesis caused by PCNB, suggesting that PCNB induced progesterone production by activating the cyclic adenosine monophosphate (cAMP/PKA) and MAPK3/1 signaling pathways. Further investigation demonstrated that PCNB induced Star expression and altered MAPK3/1 signaling in ovary tissues of immature SD rats treated with PCNB at the dose of 100, 200, or 300 mg/kg by daily gavage for 7 days, while serum progesterone level was dose-dependently decreased. We demonstrated that PCNB exposure accelerated the recruitment of primordial follicles into the growing follicle pool in ovary tissues, accompanied by increased levels of anti-Mullerian hormone (AMH) in both ovary tissues and serum. Taken together, our data demonstrate for the first time that PCNB stimulated Star expression, altered MAPK3/1 signaling and progesterone production in vivo and in vitro, and accelerated follicular development with a concomitant increase in AMH in ovary tissues and serum. Our findings provide novel insight into the toxicity of PCNB to animal ovary function.

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In vitro development of mouse fetal germ cells into mature oocytes

Reproduction ◽

10.1530/rep-06-0378 ◽

2007 ◽

Vol 134 (2) ◽

pp. 223-231 ◽

Cited By ~ 30

Author(s):

Wei Shen ◽

Lan Li ◽

Zhaodai Bai ◽

Qingjie Pan ◽

Mingxiao Ding ◽

...

Keyword(s):

Genomic Imprinting ◽

Germ Cells ◽

Follicular Development ◽

Germinal Vesicle ◽

Germinal Vesicle Breakdown ◽

Primordial Follicles ◽

Ovarian Cells ◽

Fetal Mice ◽

Vitro Development

Little is known about the mechanisms underlying primordial follicular formation and the acquisition of competence to resume meiosis by growing oocytes. It is therefore important to establish anin vitroexperimental model that allows one to study such mechanisms. Mouse follicular development has been studiedin vitroover the past several years; however, no evidence has been presented showing that mature oocytes can be obtained from mouse fetal germ cells prior to the formation of primordial follicles. In this study, a method has been established to obtain mature oocytes from the mouse fetal germ cells at 16.5 days postcoitum (dpc). From the initiation of primordial follicular formation to the growth of early secondary follicles, ovarian tissues from 16.5 dpc fetal mice were culturedin vitrofor 14 days. Subsequently, 678 intact secondary follicles were isolated from 182 mouse fetal ovaries and cultured for 12 days. A total of 141 oocytes inside antral follicles were maturedin vitro, and 102 oocytes underwent germinal vesicle breakdown. We found that 97 oocytes were fertilized and 15 embryos were able to form morula–blastocysts. We also analyzed various genomic imprinting markers and showed that the erasure of genomic imprinting markers in the parental generation was also imposed on the oocytes that developed from fetal germ cells. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with ovarian cells, and that oocytes within the growing follicles are able to mature normallyin vitro.

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190 JNK SIGNALLING IN FOLLICLE ACTIVATION

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab190 ◽

2013 ◽

Vol 25 (1) ◽

pp. 244 ◽

Cited By ~ 1

Author(s):

M. J. Bertoldo ◽

N. Duffard ◽

L. Calais ◽

C. Frapsauce ◽

D. Monniaux ◽

...

Keyword(s):

Culture Media ◽

Dose Effect ◽

Quiescent State ◽

Multinomial Regression ◽

Jnk Pathway ◽

Primordial Follicles ◽

Progesterone Synthesis ◽

Negative Factors ◽

Follicle Activation

Primordial follicles are maintained in a quiescent state until they receive a signal to activate and join the growing pool. It is essential that the rate of follicle activation is well coordinated as it determines the reproductive lifespan of the female. This finely tuned process is under the control of a variety of positive and negative factors, and there is evidence that the JNK pathway is involved. The aim of this study was to assess the effect of a JNK inhibitor (SP600125) on follicle activation in vitro. Ovaries from 6 prepubertal sheep were dissected into 1-mm3 fragments and cultured in the presence or absence of SP600125 (0, 5, or 25 µM; SP0, SP5, and SP25, respectively). After 0, 2, 5, or 9 days, fragments were fixed, sectioned, and analysed by histological morphometry to determine the number and type of follicles in addition to TUNEL analysis for apoptosis. In total, 21 584 follicles were assessed. Follicles were classified as either primordial, intermediate, primary, secondary, or antral. Culture media were also assayed for steroid content. After multinomial regression analysis, there were no differences in the rate of follicle activation between groups on Day 2 of culture. However, after 5 days of culture there were significantly more primordial follicles in SP25 (69 ± 9.15%; P < 0.01) and SP5 (52 ± 8.12%; P < 0.05) groups when compared with SP0 (34 ± 3.98%). At the end of culture (Day 9), these differences continued to be observed (SP25: 63 ± 8.47%; SP5: 51 ± 6.62%; SP0: 34 ± 5.61%; P < 0.05). On Day 9 there were more intermediate follicles following treatment with SP0 (52 ± 3.27%) compared with SP5 and SP25 (42 ± 4.72% and 31 ± 7.41% respectively; P < 0.05). There was also a tendency for more primary follicles on Day 9 for the control (11 ± 4.29%) when compared with the other treatments (4 to 7%; P = 0.06). The proportion of apoptotic primordial follicles was greatest in the SP25 group when compared with SP5 and SP0 at all timepoints (P < 0.05). After 2 days of culture, progesterone concentration was highest in the SP25 group (4.1 ± 0.45 ng mL–1) compared with the SP5 and SP0 groups (2.9 ± 0.39 ng mL–1 and 1.6 ± 0.79 ng mL–1, respectively; P < 0.05). However, this dose effect was lost by the end of culture. There was no effect of the inhibitor on oestradiol synthesis. By using SP600125, we have observed a dose-dependent block on follicle activation. These results suggest that the JNK pathway is a novel regulator of follicle activation and development. However, further confirmation is required to determine its specific action. The mechanisms by which SP25 promotes progesterone synthesis and increases apoptosis are unknown and also require clarification.

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Peroxisome Proliferator-Activated Receptor Gamma Modulator Promotes Neonatal Mouse Primordial Follicle Activation In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms21093120 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3120

Author(s):

Sook Young Yoon ◽

Ran Kim ◽

Hyunmee Jang ◽

Dong Hyuk Shin ◽

Jin Il Lee ◽

...

Keyword(s):

Embryonic Development ◽

Primordial Follicle ◽

Neonatal Mouse ◽

Peroxisome Proliferator ◽

Follicle Growth ◽

Peroxisome Proliferator Activated Receptor ◽

Primordial Follicles ◽

Primordial Follicle Activation ◽

Follicle Activation

Peroxisome proliferator-activated receptor gamma (PPARγ) is known as a regulator of cellular functions, including adipogenesis and immune cell activation. The objectives of this study were to investigate the expression of PPARγ and identify the mechanism of primordial follicle activation via PPARγ modulators in mouse ovaries. We first measured the gene expression of PPARγ and determined its relationship with phosphatase and tensin homolog (PTEN), protein kinase B (AKT1), and forkhead box O3a (FOXO3a) expression in neonatal mouse ovaries. We then incubated neonatal mouse ovaries with PPARγ modulators, including rosiglitazone (a synthetic agonist of PPARγ), GW9662 (a synthetic antagonist of PPARγ), and cyclic phosphatidic acid (cPA, a physiological inhibitor of PPARγ), followed by transplantation into adult ovariectomized mice. After the maturation of the transplanted ovaries, primordial follicle growth activation, follicle growth, and embryonic development were evaluated. Finally, the delivery of live pups after embryo transfer into recipient mice was assessed. While PPARγ was expressed in ovaries from mice of all ages, its levels were significantly increased in ovaries from 20-day-old mice. In GW9662-treated ovaries in vitro, PTEN levels were decreased, AKT was activated, and FOXO3a was excluded from the nuclei of primordial follicles. After 1 month, cPA-pretreated, transplanted ovaries produced the highest numbers of oocytes and polar bodies, exhibited the most advanced embryonic development, and had the greatest blastocyst formation rate compared to the rosiglitazone- and GW9662-pretreated groups. Additionally, the successful delivery of live pups after embryo transfer into the recipient mice transplanted with cPA-pretreated ovaries was confirmed. Our study demonstrates that PPARγ participates in primordial follicle activation and development, possibly mediated in part by the PI3K/AKT signaling pathway. Although more studies are required, adapting these findings for the activation of human primordial follicles may lead to treatments for infertility that originates from poor ovarian reserves.

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Effects of jacalin and follicle-stimulating hormone on in vitro goat primordial follicle activation, survival and gene expression

Zygote ◽

10.1017/s0967199414000173 ◽

2014 ◽

Vol 23 (4) ◽

pp. 537-549 ◽

Cited By ~ 3

Author(s):

Regislane P. Ribeiro ◽

Antonia M.L.R. Portela ◽

Anderson W.B. Silva ◽

José J.N. Costa ◽

José R.S. Passos ◽

...

Keyword(s):

Gene Expression ◽

Ovarian Tissue ◽

Follicle Stimulating Hormone ◽

Primordial Follicle ◽

Nuclear Antigen ◽

Primordial Follicles ◽

Primordial Follicle Activation ◽

Follicle Activation ◽

Stimulating Hormone

SummaryThis study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 μg/ml – Experiment 1) or in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 μg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.

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The activin-follistatin system and in vitro early follicle development in goats

Journal of Endocrinology ◽

10.1677/joe.1.06487 ◽

2006 ◽

Vol 189 (1) ◽

pp. 113-125 ◽

Cited By ~ 35

Author(s):

J R V Silva ◽

T Tharasanit ◽

M A M Taverne ◽

G C van der Weijden ◽

R R Santos ◽

...

Keyword(s):

Ovarian Tissue ◽

Primordial Follicle ◽

Activin A ◽

Terminal Deoxynucleotidyl Transferase ◽

Follicle Development ◽

Cortical Tissue ◽

Primary Follicle ◽

Growth And Survival ◽

Primordial Follicles

The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.

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Transcriptomic profiling of neonatal mouse granulosa cells reveals new insights into primordial follicle activation

Biology of Reproduction ◽

10.1093/biolre/ioab193 ◽

2021 ◽

Author(s):

Emmalee A Ford ◽

Emily R Frost ◽

Emma L Beckett ◽

Shaun D Roman ◽

Eileen A McLaughlin ◽

...

Keyword(s):

Gene Expression ◽

Granulosa Cells ◽

Expression Profiles ◽

Primordial Follicle ◽

Differentially Expressed ◽

Primary Follicle ◽

Primordial Follicles ◽

Time Points ◽

Primordial Follicle Activation ◽

Follicle Activation

Abstract The dormant population of ovarian primordial follicles is determined at birth and serves as the reservoir for future female fertility. Yet our understanding of the molecular, biochemical, and cellular processes underpinning primordial follicle activation remains limited. The survival of primordial follicles relies on the correct complement and morphology of granulosa cells, which provide signalling factors essential for oocyte and follicular survival. To investigate the contribution of granulosa cells in the primordial-to-primary follicle transition, gene expression profiles of granulosa cells undergoing early differentiation were assessed in a murine model. Ovaries from C57Bl/6 mice were enzymatically dissociated at time-points spanning the initial wave of primordial follicle activation. Post-natal day (PND) 1 ovaries yielded primordial granulosa cells, and PND4 ovaries yielded a mixed population of primordial and primary granulosa cells. The comparative transcriptome of granulosa cells at these time-points was generated via Illumina NextSeq 500 system which identified 131 significantly differentially expressed transcripts. The differential expression of eight of the transcripts was confirmed by RT-qPCR Following biological network mapping via Ingenuity Pathway Analysis, the functional expression of the protein products of three of the differentially expressed genes, namely FRZB, POD1 and ZFX, was investigated with in-situ immunolocalisation in PND4 mouse ovaries was investigated. Finally, evidence was provided that Wnt pathway antagonist, secreted frizzled-related protein 3 (FRZB), interacts with a suppressor of primordial follicle activation WNT3A and may be involved in promoting primordial follicle activation. This study highlights the dynamic changes in gene expression of granulosa cells during primordial follicle activation and provides evidence for a renewed focus into the Wnt signalling pathway’s role in primordial follicle activation.

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