Development Of Primary Follicles Research Articles

Human mesenchymal stem cells derived exosomes improve ovarian function in chemotherapy-induced premature ovarian insufficiency mice by inhibiting ferroptosis through Nrf2/GPX4 pathway.

Chemotherapy exposure has become a main cause of premature ovarian insufficiency (POI). This study aimed to evaluate the role and molecular mechanism of human umbilical cord mesenchymal stem cell-derived exosomes (hUMSC-Exos) in ovarian function protection after chemotherapy. hUMSC-Exos were applied to cyclophosphamide-induced premature ovarian insufficiency mice and human ovarian granulosa tumor cells (KGN) to determine their effects on follicular development and granulosa cell apoptosis. Evaluation was done for iron ion and reactive oxygen species (ROS) production, lipid peroxidation levels, and changes in iron death-related molecules (nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Glutathione Peroxidase enzyme 4 (GPX4), and Solute carrier family 7 member 11 cystine glutamate transporter (SLC7A11; xCT)). Furthermore, rescue experiments using an Nrf2 inhibitor were performed to assess the therapeutic effects of hUMSC-Exos on granulosa cells. hUMSC-Exos promoted ovarian hormone levels and primary follicle development in POI mice and reduced granulosa cell apoptosis. After hUMSC-Exos treatment, the ROS production, free iron ions and lipid peroxidation levels of granulosa cells decreased, and the iron death marker proteins Nrf2, xCT and GPX4 also decreased. Furthermore, the Nrf2 inhibitor ML385 significantly attenuated the effects of hUMSC-Exos on granulosa cells. hUMSC-Exos inhibit ferroptosis and protect against CTX-induced ovarian damage and granulosa cell apoptosis through the Nrf2/GPX4 signaling pathway, revealing a novel mechanism of hUMSC-Exos in POI therapy.

SRSF1 is essential for primary follicle development by regulating granulosa cell survival via mRNA alternative splicing.

Granulosa cell abnormalities are characteristics of premature ovarian insufficiency (POI). Abnormal expression of serine/arginine-rich splicing factor 1 (SRSF1) can cause various diseases, but the role of SRSF1 in mouse granulosa cells remains largely unclear. In this study, we found that SRSF1 was expressed in the nuclei of both mouse oocytes and granulosa cells. The specific knockout of Srsf1 in granulosa cells led to follicular development inhibition, decreased granulosa cell proliferation, and increased apoptosis. Gene Ontology (GO) analysis of RNA-seq results revealed abnormal expression of genes involved in DNA repair, cell killing and other signalling pathways. Alternative splicing (AS) analysis showed that SRSF1 affected DNA damage in granulosa cells by regulating genes related to DNA repair. In summary, SRSF1 in granulosa cells controls follicular development by regulating AS of genes associated with DNA repair, thereby affecting female reproduction.

Mouse primary follicles experience slow growth rates after activation and progressive increases that influence the duration of the primary follicle phase†.

There are conflicting estimates of the duration of mouse primary follicle development. An accurate determination is needed for studies examining preantral follicle survival and mathematical modeling of folliculogenesis. Primary follicle granulosa cell proliferation rates are low and variable, which may explain the variation in duration estimates. In the present study, female C57Bl6/J mice were exposed to bromodeoxyuridine for 48hours, to label the proliferating granulosa cells in a large proportion of primary follicles. The bromodeoxyuridine-containing water was then withdrawn and replaced with drug-free water and the mice were euthanized at 0, 1, 3, 6, 10, or 13days post-bromodeoxyuridine withdrawal. Granulosa cells were bromodeoxyuridine labeled in 48% of primary follicles at day 0, but this decreased to 5% over the 13-day period, as the labeled primary follicles progressed to the secondary follicle stage. Curve-fitting estimated that the last of the bromodeoxyuridine-labeled primary follicles would progress to the secondary stage by 13.7days. Mathematical models that assumed constant rates of primary follicle proliferation were fitted to the data, but the observed pattern of bromodeoxyuridine-labeled primary follicle disappearance could not be replicated. The level of immunoreactivity for bromodeoxyuridine and proliferating-cell nuclear antigen in primary follicles revealed follicles with no granulosa cell proliferation during the 48-h bromodeoxyuridine-exposure period had resumed proliferation 1 or 3days later. Therefore, primary follicle granulosa cells proliferate after follicle activation, but proliferation rates gradually increase as the follicle develops. Prior estimates of primary follicle duration are inaccurate due to the assumption that follicles develop at a constant rate.

A modified hydrostatic microfluidic pumpless device for in vitro murine ovarian tissue culture as research model for fertility preservation.

This study aimed to compare the efficacies of conventional and non-conventional (modified hydrostatic microfluidic pumpless device, MHPD) systems on ovarian tissue culture and in vitro follicle growth using a mouse model. A total of 56 ovarian cortical tissues retrieved from seven wild-type mice were divided into three groups: 1) fresh control, 2) conventional culture system (control), and 3) non-conventional system with MHPD. Ovarian tissues were cultured for 96 hours and evaluated for follicle morphology, developmental stage, intact follicle density, and relative gene expression levels (proliferating cell nuclear antigen, insulin like growth factor 1, BAX, and Bcl-2). Our major data demonstrated that the mean percentage of primary follicle development was increased by the MHPD (P<0.05). In addition, this device could maintain and support follicle development better than the conventional culture systems. However, the overall outcomes were not significantly improved by our first-design prototype. Consequently, next-generation platforms should be developed as alternative medical tools for fertility preservation research.

Proteomic Analysis Identifies Potential Markers for Chicken Primary Follicle Development.

Simple SummaryOur study presents a comprehensive approach elaborating the mechanism of primary follicle development in the chicken. The identified differentially expressed proteins of small and developing primary follicles (SPFs and DPFs) could be used as potential markers in chicken primary follicle development. The DEPs have their functional involvement in different processes including glycolysis, pyruvate metabolism, amino acid synthesis, and oocyte meiosis. The Anxa2, Pdia3, and Capzb have a connotation in primary follicle development. These findings were validated by real-time quantitative PCR and provided a basis for the exploration of DEPs as suitable makers related to the primary follicle development in chicken.Follicles’ development in chicken imparts a major impact on egg production. To enhance the egg-laying efficiency, comprehensive knowledge of different phases of follicular development is a prerequisite. Therefore, we used the tandem mass tag (TMT) based proteomic approach to find the genes involved in the primary follicular development of chicken. The primary follicles were divided into two groups—small primary follicles (81–150 μm) and developed primary follicles (300–500 μm). Differential expression analysis (fold change > 1.2, p-value < 0.05) revealed a total of 70 differentially expressed proteins (DEPs), of which 38 were upregulated and 32 were downregulated. Gene ontology (GO) enrichment analysis disclosed that DEPs were intricate with cellular protein localization, the establishment of protein localization, and nucleoside phosphate-binding activities. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway indicated the involvement of DEPs in different metabolic pathways such as glycolysis, pyruvate metabolism, galactose metabolism, and fructose and mannose metabolism. The current proteomic analysis suggested suitable markers such as Anxa2, Pdia3, and Capzb, which may serve as a potential role for primary follicle development. The present study provides the first insight into the proteome dynamics of primary follicle development and would play a potential role for further studies in chicken to improve egg productivity.

Histopathological alterations of male and female reproductive systems induced by alloxan in rats

The objective of this study is to determine the histological effects experimentally induced by injection of alloxan 100 mg/kg B.w. on the histopathological structure of reproductive organs of male and female albino rats. The results showed that treatment with alloxan cause alteration in testis include irregular shape and size of seminiferous tubules, irregular division of spermatid cells, degeneration and necrosis of Sertoli cells and paucity of sperms in the lumen of tubules. While histological examination of epididymis showed the lumen of it free from sperms, thickening of muscular layer and interstitial tissue between the epididymis canal. The histological alteration of female reproductive organs includes disturbances in development of primary follicles of ovaries, hemorrhage in the interstitial tissue as well as atrophy in the uterine glands with hyperplasia of the epithelial cells of uterus. The conclusion of this study showed that alloxan cause histological alteration in reproductive organs of male and female rats.

Molecular action of pyriproxyfen: Role of the Methoprene-tolerant protein in the pyriproxyfen-induced sterilization of adult female mosquitoes.

Exposure of adult mosquitoes to pyriproxyfen (PPF), an analog of insect juvenile hormone (JH), has shown promise to effectively sterilize female mosquitoes. However, the underlying mechanisms of the PPF-induced decrease in mosquito fecundity are largely unknown. We performed a comprehensive study to dissect the mode of PPF action in Aedes aegypti mosquitoes. Exposure to PPF prompted the overgrowth of primary follicles in sugar-fed Ae. aegypti females but blocked the development of primary follicles at Christopher’s Stage III after blood feeding. Secondary follicles were precociously activated in PPF-treated mosquitoes. Moreover, PPF substantially altered the expression of many genes that are essential for mosquito physiology and oocyte development in the fat body and ovary. In particular, many metabolic genes were differentially expressed in response to PPF treatment, thereby affecting the mobilization and utilization of energy reserves. Furthermore, PPF treatment on the previtellogenic female adults considerably modified mosquito responses to JH and 20-hydroxyecdysone (20E), two major hormones that govern mosquito reproduction. Krüppel homolog 1, a JH-inducible transcriptional regulator, showed consistently elevated expression after PPF exposure. Conversely, PPF upregulated the expression of several key players of the 20E regulatory cascades, including HR3 and E75A, in the previtellogenic stage. After blood-feeding, the expression of these 20E response genes was significantly weaker in PPF-treated mosquitoes than the solvent-treated control groups. RNAi-mediated knockdown of the Methoprene-tolerant (Met) protein, the JH receptor, partially rescued the impaired follicular development after PPF exposure and substantially increased the hatching of the eggs produced by PPF-treated female mosquitoes. Thus, the results suggested that PPF relied on Met to exert its sterilizing effects on female mosquitoes. In summary, this study finds that PPF exposure disturbs normal hormonal responses and metabolism in Ae. aegypti, shedding light on the molecular targets and the downstream signaling pathways activated by PPF.

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.

Aneuploidy in dizygotic twin sheep detected using genome-wide single nucleotide polymorphism data from two commonly used commercial vendors

Early detection of karyotype abnormalities, including aneuploidy, could aid producers in identifying animals which, for example, would not be suitable candidate parents. Genome-wide genetic marker data in the form of single nucleotide polymorphisms (SNPs) are now being routinely generated on animals. The objective of the present study was to describe the statistics that could be generated from the allele intensity values from such SNP data to diagnose karyotype abnormalities; of particular interest was whether detection of aneuploidy was possible with both commonly used genotyping platforms in agricultural species, namely the Applied BiosystemsTM AxiomTM and the Illumina platform. The hypothesis was tested using a case study of a set of dizygotic X-chromosome monosomy 53,X sheep twins. Genome-wide SNP data were available from the Illumina platform (11 082 autosomal and 191 X-chromosome SNPs) on 1848 male and 8954 female sheep and available from the AxiomTM platform (11 128 autosomal and 68 X-chromosome SNPs) on 383 female sheep. Genotype allele intensity values, either as their original raw values or transformed to logarithm intensity ratio (LRR), were used to accurately diagnose two dizygotic (i.e. fraternal) twin 53,X sheep, both of which received their single X chromosome from their sire. This is the first reported case of 53,X dizygotic twins in any species. Relative to the X-chromosome SNP genotype mean allele intensity values of normal females, the mean allele intensity value of SNP genotypes on the X chromosome of the two females monosomic for the X chromosome was 7.45 to 12.4 standard deviations less, and were easily detectable using either the AxiomTM or Illumina genotype platform; the next lowest mean allele intensity value of a female was 4.71 or 3.3 standard deviations less than the population mean depending on the platform used. Both 53,X females could also be detected based on the genotype LRR although this was more easily detectable when comparing the mean LRR of the X chromosome of each female to the mean LRR of their respective autosomes. On autopsy, the ovaries of the two sheep were small for their age and evidence of prior ovulation was not appreciated. In both sheep, the density of primordial follicles in the ovarian cortex was lower than normally found in ovine ovaries and primary follicle development was not observed. Mammary gland development was very limited. Results substantiate previous studies in other species that aneuploidy can be readily detected using SNP genotype allele intensity values generally already available, and the approach proposed in the present study was agnostic to genotype platform.

Effects of Fatty Acid and Geraniol Repellent-Oil Mixtures Applied to Cattle on Blood Feeding and Reproductive Parameters in Field Populations of Haematobia irritans (Diptera: Muscidae).

California pastured cattle were treated with 250 ml of a 15% mixture of fatty acids (C8-C9-C10) or 125 ml of 2% geraniol in a mineral oil carrier to assess impacts on horn flies, Haematobia irritans (L.) (Diptera: Muscidae) over two summers. Horn flies were netted from cattle every 3-4 d for 2 wk before treatment, 2 wk during treatment (four treatments, with flies collected before each treatment), and 2 wk after treatments ceased. Blood meal weights were estimated by hemoglobin assay of excised abdomens. Other females were dissected to determine the number of active ovarioles and the stage of primary follicle development. Depending on year and herd, pretreatment males contained an average of 0.6-1.0 mg of blood, while females contained 1.7-2.7 mg. Pretreatment egg development (least developed oocytes were stage 1 and fully developed eggs were stage 5) averaged 3.7-4.3, and number of active ovarioles averaged 18.1 to 19.6/female. During treatment periods, significant reductions in blood weight were noted for females, but usually not for males, and females also often exhibited reduced mean oocyte stage and number of active ovarioles. Peaks in proportions of young nulliparous females (oocyte stages 1 or 2) were seen during some repellent application periods. This suggested older females had been killed or driven off from the local population by the treatments, and flies on cattle included more young flies that likely were recent arrivals. The repellent-oil mixture thus impacted blood feeding, reproductive fitness, and probably age structure in the field.

A teleost androgen promotes development of primary ovarian follicles in coho salmon and rapidly alters the ovarian transcriptome.

Recent studies using several teleost models have revealed that androgens increase the size of previtellogenic (primary and/or early secondary) ovarian follicles. To explore our hypothesis that androgens drive the development of primary follicles into early secondary follicles, and to determine the mechanisms underlying these androgenic effects, we exposed juvenile coho salmon to near-physiological and relatively sustained levels of the nonaromatizable androgen 11-ketotestosterone (11-KT). This resulted in significant growth of primary ovarian follicles after 10 and 20 days, with follicles after 20 days displaying a morphological phenotype characteristic of early secondary follicles (presence of cortical alveoli). Utilizing the same experimental approach, we then analyzed how 11-KT rapidly altered the ovarian transcriptome after 1 and 3 days of treatment. RNA-Seq analysis revealed that 69 (day 1) and 1,022 (day 3) contiguous sequences (contigs) were differentially expressed relative to controls. The differentially expressed contigs mapped to genes including those encoding proteins involved in gonadotropin, steroid hormone, and growth factor signaling, and in cell and ovarian development, including genes with putative androgen-response elements. Biological functions and canonical pathways identified as potentially altered by 11-KT include those involved in ovarian development, tissue differentiation and remodeling, and lipid metabolism. We conclude that androgens play a major role in stimulating primary ovarian follicle development and the transition into secondary growth.

Association between BMP15 Gene Polymorphism and Reproduction Traits and Its Tissues Expression Characteristics in Chicken

BMP15 (Bone morphogenetic protein 15) is an oocyte-secreted growth factor required for ovarian follicle development and ovulation in mammals, but its effects on reproduction in chickens are unclear. In this study, the association between BMP15 polymorphisms and reproduction traits were analyzed, and its expression characteristics in different tissues were explored in LaiWu Black chickens. Three single nucleotide polymorphisms (SNPs) were identified in four hundred LaiWu Black chickens. One SNP (NC_006091.3:g.1773T>C) located in exon 2 which was significantly associated with egg weight at first egg (EWFE) (P = 0.0389), was novel. Diplotypes based on the three SNPs were found to be significantly associated with egg weight at age of 43W (EW43) (P = 0.0058). The chickens with H3H3 diplotype had their first egg 0.57 days later than chickens with H5H5 diplotype and 1.21 days-3.96 days earlier than the other five diplotype chickens. The egg production at age of 43W (E43), egg production at age of 46W (E46) and egg production at age of 48W (E48) for chickens with H3H3 diplotype were the highest among all the chickens, and the E48 of chickens with H3H3 diplotype had 11.83 eggs higher than chickens with H1H5 diplotype. RT-qPCR results showed that the expression level of BMP15 gene in ovarian follicle was in the order of 4 mm>6 mm -8 mm> 15 mm -19 mm> 23 mm -29 mm > 33 mm -34 mm in diameter. The mRNA level in follicles of 4 mm and 6–8 mm in diameter were significantly higher than that in the other follicles (P<0.01). In the same week, the highest mRNA level was found in the ovary, and it was significantly different from that found in the liver and oviduct (P<0.01). Our results indicate that BMP15 plays a vital role in the development of ovary and follicles, especially in the development of primary follicles. H3H3 may be an potential advantageous molecular marker for improving reproduction traits in chickens.

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.

Oocyte-derived R-spondin2 promotes ovarian follicle development.

R-spondin proteins are adult stem cell growth factors capable of stimulating gut development by activating LGR4, 5, and 6 receptors to promote Wnt signaling. Although multiple Wnt ligands and cognate Frizzled receptors are expressed in the ovary, their physiological roles are unclear. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R-spondin2 in oocytes of ovarian follicles. In cultured somatic cells from preantral follicles, R-spondin2 treatment (ED50: 3 ng/ml) synergized with Wnt3a to stimulate Wnt signaling. In cultured ovarian explants from prepubertal mice containing preantral follicles, treatment with R-spondin2, similar to follicle stimulating hormone, promoted the development of primary follicles to the secondary stage. In vivo administration of an R-spondin agonist stimulated the development of primary follicles to the antral stage in both immature mice and gonadotropin releasing hormone antagonist-treated adult mice. Subsequent treatment with gonadotropins allowed the generation of mature oocytes capable of undergoing early embryonic development and successful pregnancy. Furthermore, R-spondin agonist treatment of immune-deficient mice grafted with human cortical fragments stimulated the development of primary follicles to the secondary stage. Thus, oocyte-derived R-spondin2 is a paracrine factor essential for primary follicle development, and R-spondin agonists could provide a new treatment regimen for infertile women with low responses to the traditional gonadotropin therapy.

123. ACTIVIN A AND OVARIAN FOLLICLE DEVELOPMENT

A significant developmental stage in ovarian folliculogenesis is the acquisition of gonadotropin sensitivity by ovarian follicles. Activin has previously been suggested to be involved in the responsiveness of granulosa cells to FSH (1). Therefore, the role of activin was investigated using a ‘physiological’ culture system to determine if pathways exist to transduce activin signals within the postnatal rat ovary. Organ cultures with day 4 whole ovaries were employed in order to assess the potential impact of Activin A on follicle growth and transition from the primordial through to the primary and later preantral stages of development. Ovaries were isolated and cultured for 10 days with the addition of supplemented DMEM/Hams F-12 media (2)and either FSH (100ng/ml), Activin A (50ng/ml), or a combination of the two. Media and treatments were refreshed every alternate day. At the end of the culture period, ovaries were fixed and sectioned, or placed immediately into Ultraspec for RNA extraction for future real-time PCR. Sections were used for morphological assessment and ovarian follicle counting of primordial, primary and preantral follicles. An evaluation of atresia by the detection of apoptotic cells was undertaken using terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick-end labeling (TUNEL). Primary follicle numbers increased significantly (P&lt;0.05) in the combined treatment group whereas, preantral follicle numbers increased significantly (P&lt;0.0001) when treated with Activin A alone. This is consistent with a morphological appraisal of atresia where a decrease in atresia was found in primordial and primary follicles, supporting the primary follicle development data and Activin A treatment alone resulted in more healthy primary and preantral follicles than atretic ones. Therefore, a stimulatory role for Activin A both in the presence of FSH (primary follicle development) or alone (preantral follicle development) has resulted in more follicles making the transition from the primordial to primary stages, as well as to the later preantral stages.

In vitro growth and differentiation of primary follicles isolated from cryopreserved sheep ovarian tissue

Achieving full in vitro growth of oocytes of both domestic animals and humans remains a major challenge. The objective of this study was to examine the in vitro development of primary follicles isolated enzymatically from cryopreserved sheep ovarian tissue. In Experiment 1, isolated primary follicles (mean diameter 60.1 ± 0.78 μm) were cultured in serum-free medium on fibronectin-coated wells for 42 days. Initially follicular structure was lost as granulosa cells plated down, but by Day 7 two distinct morphologies began to emerge. Nineteen out of 36 oocytes were gradually re-surrounded by granulosa cells, forming follicle-like units (reorganized follicles), and the remaining 17 were not (non-reorganized follicles). On Day 2, there was no difference in diameter of oocytes between reorganized and non-reorganized follicles. The diameter (mean ± S.E.M.) of oocytes of reorganized follicles increased ( P < 0.05) from 47.1 ± 2.2 μm to 65.3 ± 2.6 μm between Day 2 and Day 42, respectively, but that of oocytes of non-reorganized follicles showed no change. In Experiment 2, oocyte growth and granulosa cell differentiation during long-term culture of primary follicles (>42 days) were examined. Oocytes of reorganized follicles reached a maximum diameter of 75.4 ± 2.0 μm, a size equivalent to that of oocytes of ovine secondary follicles. Using RT-PCR, mRNA for follicle stimulating hormone receptor was detected in granulosa cells of freshly isolated secondary follicles and of long-term cultured reorganized follicles, but not of non-reorganized follicles. In Experiment 3, we tested if the culture conditions could support further oocyte growth in secondary follicles. The oocytes from enzymatically isolated secondary follicles increased in diameter from 77.7 ± 1.6 μm to 98.8 ± 2.1 μm ( P < 0.05) during 28 days in culture. The changes in oocyte size and in gene expression by granulosa cells support the conclusion that isolated ovine primary follicles developed in vitro to reach the secondary follicle stage.

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

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.

Regulation of ovarian primordial follicle assembly and development by estrogen and progesterone: endocrine model of follicle assembly.

The assembly of the developmentally arrested primordial follicle and the subsequent transition of the primordial follicle to the primary follicle are critical processes in normal ovarian physiology that remain to be elucidated. Ovarian follicles do not proliferate and the primordial follicles present in the neonate represent the total number of gametes available to a female throughout her reproductive life. The primordial follicles are oocytes surrounded by less differentiated squamous granulosa cells and are derived from oocyte nests, and primary follicles are oocytes surrounded by a single layer of cuboidal granulosa cells that have initiated follicle development. Abnormalities in primordial follicle assembly, arrest, and development (i.e. primordial to primary follicle transition) can cause pathological conditions such as premature ovarian failure. In this study newborn rat ovaries were cultured for 7 d. The rate of primordial follicle assembly in vivo was identical with the rate in vitro. Interestingly, the rate of primordial follicle transition to the primary follicle was found to be 3 times greater in culture. This abnormal rate of primary follicle development in culture suggests the primordial follicle does not arrest in development as observed in vivo. To investigate this phenomena newborn rat ovaries were cultured in the presence of progesterone, estradiol or calf serum. Estradiol, progesterone, or calf serum significantly reduced the level of initial primordial to primary follicle transition. Approximately 60% of follicles make the primordial to primary follicle transition in control ovaries and about 30% in treated ovaries. Steroids and calf serum had no effect on the primordial to primary follicle transition in ovaries collected and cultured from postnatal 4-d-old rats, suggesting the effects observed are restricted to the initial wave of primordial to primary follicle transition. Interestingly, progesterone was also found to significantly reduce the rate of primordial follicle assembly. All viable oocytes assembled into primordial follicles in control ovaries and approximately 40% remained unassembled in progesterone-treated ovaries. Progesterone was also found to reduce primordial follicle assembly in vivo with 10% of the total follicles remaining unassembled in progesterone injected neonatal animals. Analysis of cellular apoptosis demonstrated that progesterone inhibited the coordinated oocyte apoptosis required for primordial follicle assembly. The hypothesis developed is that high levels of maternal and fetal steroids prevent premature primordial follicle assembly and primordial to primary follicle transition in the embryo. After birth steroid levels fall dramatically and the primordial follicles are free to assemble and initiate development. These observations suggest a novel role for steroids and the maternal-fetal endocrine unit in the control of ovarian primordial follicle assembly and early follicular development.

Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development.

The ovary contains a pool of primordial follicles containing oocytes arrested in meiosis that are the source of developing follicles for the female. Growth and differentiation factor-9 (GDF-9) is a member of the transforming growth factor beta superfamily of growth factors, and follicles of GDF-9 knockout mice arrest in the primary stage of development. The effect of GDF-9 treatment on the primordial to primary follicle transition and on subsequent follicle progression was examined using a rat ovary organ culture system. Ovaries from 4-day-old rats were cultured under serum-free conditions in the absence or presence of growth factors. GDF-9 treatment caused a decrease in the proportion of stage 1 early primary follicles and a concomitant increase in the proportion of stage 2 mature primary follicles. GDF-9 did not effect primordial follicles or stage 0 to stage 1 follicle transition. GDF-9 also did not influence stage 3 or 4 secondary follicle numbers. Isolated antral follicle granulosa and theca cell cultures were used to analyze the actions of GDF-9. GDF-9 treatment did not directly influence either granulosa or theca cell proliferation. The ability of GDF-9 to influence the expression of another growth factor was examined. GDF-9 treatment increased kit ligand (KL) mRNA expression in bovine granulosa cells after 2 days of culture. Ovaries from 4-day-old rats were also cultured with or without GDF-9 treatment, and total ovary expression of KL mRNA was increased by GDF-9. In summary, GDF-9 was found to promote the progression of early primary follicle development but did not influence primordial follicle development. The actions of GDF-9 on specific stages of follicle development may in part be mediated through altering the expression of KL.

Effect of growth factors and CO-culture with ovarian medulla on the activation of primordial follicles in explants of bovine ovarian cortex

It has been proposed that the ovarian medulla exerts an intra-ovarian inhibitory effect on primordial follicle activation in cattle. We tested this hypothesis using cortical ovarian explants and determined whether growth factors could alter follicle activation or primary follicle health. Ovaries were obtained from bovine fetuses, and cortical slices were cultured on Millicell culture inserts for up to 8 days. Within 2 d of culture, the proportion of primordial follicles decreased from 70.1±3.5 to 6.4±3.4% (P<0.05), and the proportion of primary follicles increased from 23.8±3.3 to 79.7+5.5% (P<0.05). The proportion of secondary follicles was relatively stable (6 to 13%). Morphological examination indicated that 91.9±3.7, 76.7±8.8, and 71.8±10.4% of primordial, primary, and secondary follicles, respectively, were considered to be healthy in slices of fresh tissue; these proportions were not altered by up to 8 d of culture (P>0.05). The proportion of all classes of follicles and their morphological health were not affected by the addition of medullary slices to the culture well, nor by the culture of cortico-medullary slices (P>0.05). The addition of FSH, insulin-like growth factor-I, epidermal growth factor, basic fibroblast growth factor, or transforming growth factor-β did not alter primordial follicle activation or the morphological health of primary or secondary follicles. The addition of transforming growth factor-α (TGFα) decreased the proportion of primary follicles that were healthy from 67.6±5.1 to 36.8±4.7% (P<0.05). In conclusion, these data do not support the existence of a medullary inhibitor of primary follicle activation but suggest a role for TGFα in the regulation of primary follicle development.