Primordial Follicle

Order of changes in ovary.1 - Menstruation2 - Developing follicle3 - Mature follicle4 - Ovulation5 - Corpus luteum6 - Deterioration of corpus luteumIn, folliculogenesis is the maturation of the, a densely packed shell of that contains an immature. Folliculogenesis describes the progression of a number of small into large that occurs in part during the.Contrary to male, which can last indefinitely, folliculogenesis ends when the remaining follicles in the are incapable of responding to the hormonal cues that previously recruited some follicles to mature. This depletion in follicle supply signals the beginning of.

Primordial follicle. Primordial germ cells migrate into the developing gonad early in embryogenesis, and differentiate into oogonia. These oogonia proliferate by mitosis. Some of these enlarge and develop into larger cells called primary oocytes and enter the first meiotic division on the pathway to making gametes by meiosis.

Contents.Overview The primary role of the follicle is support. From birth, the of the human female contain a number of immature,. These follicles each contain a similarly immature. At clutches of follicles begin folliculogenesis, entering a growth pattern that ends in death (apoptosis) or in ovulation (the process where the oocyte leaves the follicle).During follicular development, primordial follicles undergo a series of critical changes in character, both histologically and hormonally. First they change into primary follicles and later into secondary follicles. The follicles then transition to.

At this stage in development, they become dependent on hormones, particularly FSH which causes a substantial increase in their growth rate. Ruptures and discharges the oocyte (that has become a ), ending folliculogenesis. Diagram of folliculogenesis, starting from pre-antral (late secondary), courtesy NCBI Phases of development Folliculogenesis is continuous, meaning that at any time the ovary contains follicles in many stages of development.

The majority of follicles die and never complete development. A few develop fully to produce a secondary oocyte which is released by rupture of the follicle in a process called.The growing follicle passes through the following distinct stages that are defined by certain structural characteristics:In a larger perspective, the whole folliculogenesis, from primordial to preovulatory follicle, belongs to the stage of of. Further information: StageDescriptionSizePrimordial, small, only one layer of flat cellsPrimordial follicles are about 0.03-0.05 mm in diameter.PrimaryMitotic cells, granulosa cellsAlmost 0.1 mm in diameterSecondaryPresence of, multiple layers of granulosa cellsThe follicle is now 0.2 mm in diameterEarly tertiaryThe early tertiary follicle is arbitrarily divided into five classes. Class 1 follicles are 0.2 mm in diameter, class 2 about 0.4 mm, class 3 about 0.9 mm, class 4 about 2 mm, and class 5 about 5 mm.Late tertiaryFully formed, no further cytodifferentiation, no novel progressClass 6 follicles are about 10 mm in diameter, class 7 about 16 mm, and class 8 about 20 mm.

It is common for non-dominant follicles to grow beyond class 5, but rarely is there more than one class 8 follicle.PreovulatoryBuilding growth in estrogen concentration, all other follicles or deadIn addition, follicles that have formed an are called or Graafian follicles. Definitions differ in where this shift occurs in the staging given above, with some stating that it occurs when entering the secondary stage, and others stating that it occurs when entering the tertiary stage.Until the preovulatory stage, the follicle contains a primary oocyte that is arrested in prophase of. During the late preovulatory stage, the oocyte continues meiosis and becomes a secondary oocyte, arrested in. (a) The maturation of a follicle is shown in a clockwise direction proceeding from the primordial follicles.

FSH stimulates the growth of a tertiary follicle, and LH stimulates the production of estrogen by granulosa and theca cells. Once the follicle is mature, it ruptures and releases the oocyte. Cells remaining in the follicle then develop into the corpus luteum. (b) In this electron micrograph of a secondary follicle, the oocyte, theca cells (thecae folliculi), and developing antrum are clearly visible. Electron microscopy images Primordial At 18–22 weeks post-conception, the cortex of the female (foetal female ovary) contains its peak number of follicles (about 4 to 5 million in the average case, but individual peak populations range from 6 to 7 million). These primordial follicles contain immature oocytes surrounded by flat, squamous (support cells) that are segregated from the oocyte's environment by the basal lamina. They are quiescent, showing little to no.

Because primordial follicles can be dormant for up to 50 years in the human, the length of the ovarian cycle does not include this time.The supply of follicles decreases slightly before birth, and to 180,000 by puberty for the average case (populations at puberty range from 25,000 to 1.5 million). By virtue of the 'inefficient' nature of folliculogenesis (discussed later), only 400 of these follicles will ever reach the preovulatory stage. At, only 1,000 follicles remain. It seems likely that early menopause occurs for women with low populations at birth, and late menopause occurs for women with high populations at birth, but there is as yet no clinical evidence for this.The process by which primordial cells 'wake up' is known as initial recruitment. Research has shown that initial recruitment is mediated by the counterbalance of various stimulatory and inhibitory hormones and locally produced growth factors. Primary During, the granulosa cells of the primordial follicles change from a flat to a cuboidal structure, marking the beginning of the primary follicle. The oocyte genome is activated and genes become.

Rudimentary pathways that are vital for communication between the follicle and oocyte are formed. Both the oocyte and the follicle grow dramatically, increasing to almost 0.1 mm in diameter.Primary follicles develop receptors to (FSH) at this time, but they are gonadotropin-independent until the antral stage. Research has shown, however, that the presence of FSH accelerates follicle growth in vitro.A glycoprotein polymer capsule called the forms around the oocyte, separating it from the surrounding granulosa cells. The zona pellucida, which remains with the oocyte after ovulation, contains enzymes that catalyze with to allow penetration.Secondary Stroma-like theca cells are recruited by oocyte-secreted signals.

They surround the follicle's outermost layer, the, and undergo cytodifferentiation to become the. An intricate network of capillary vessels forms between these two thecal layers and begins to circulate blood to and from the follicle.The late-term secondary follicle is marked histologically and structurally by a fully grown oocyte surrounded by a zona pellucida, approximately nine layers of granulosa cells, a basal lamina, a theca interna, a capillary net, and a theca externa. The development of the antrum also starts taking place in secondary follicle stageAntrum formation.

Further information:The formation of a fluid-filled cavity adjacent to the oocyte called the designates the follicle as an, in contrast to a so-called preantral follicle that still lacks an antrum. An antral follicle is also called a Graafian follicle.Definitions differ as to which stage this shift occurs in, with some designating follicles in the secondary stage as antral, and others designating them as preantral. Early tertiary In the tertiary follicle, the basic structure of the mature follicle has formed and no novel cells are detectable. Granulosa and theca cells continue to undergo mitotis concomitant with an increase in antrum volume.

Tertiary follicles can attain a tremendous size that is hampered only by the availability of FSH, which it is now dependent on.Under action of an oocyte-secreted morphogenic gradient, the granulosa cells of the tertiary follicle undergo differentiation into four distinct subtypes: corona radiata, surrounding the zona pellucida; membrana, interior to the basal lamina; periantral, adjacent to the antrum and cumulus oophorous, which connects the membrana and corona radiata granulosa cells together. Each type of cell behaves differently in response to FSH.Theca interna cells express receptors for luteinizing hormone.

LH induces the production of by the theca cells, most notably, which are aromatized by granulosa cells to produce, primarily. Consequently, estrogen levels begin to rise.Late tertiary and preovulatory (the follicular phase of the menstrual cycle) At this point, the majority of the group of follicles that started growth have died. This process of follicle death is known as, and it is characterized by radical of all constituent cells and the oocyte. Although it is not known what causes atresia, the presence of high concentrations of FSH has been shown to prevent it.A rise in pituitary FSH caused by the disintegration of the corpus luteum at the conclusion of a menstrual cycle precipitates the recruitment of five to seven class 5 follicles to participate in the next cycle. These follicles enter the end of the prior menstrual cycle and transition into the of the next one.

The selected follicles, called antral follicles, compete with each other for growth-inducing FSH.The pattern of this emergence of a cohort of five to seven antral follicles is debated. There are theories of continuous recruitment of antral follicles, theories of a single recruitment episode at the end of the luteal phase, and more recently there has been evidence for a recruitment model marked by 2 - 3 waves of follicle recruitment and development during the menstrual cycle (only one of which is actually an ovulatory wave).In response to the rise of FSH, the antral follicles begin to secrete estrogen and, which have a negative feedback effect on FSH. Follicles that have fewer will not be able to develop further; they will show retardation of their growth rate and become atretic. Eventually, only one follicle will be viable. This remaining follicle, called the dominant follicle, will grow quickly and dramatically—up to 20 mm in diameter—to become the preovulatory follicle.Note: Many sources misrepresent the pace of follicle growth, some even suggesting that it takes only fourteen days for a primordial follicle to become preovulatory. Actually, the follicular phase of the menstrual cycle means the time between selection of a tertiary follicle and its subsequent growth into a preovulatory follicle.

The actual time for development of a follicle varies.The growth of the dominant follicle during the is about 1.5 mm per day (±0.1 mm), both in natural cycles and for any dominant follicle developing while taking. Performing leads to a greater recruitment of follicles, growing at about 1.6 mm per day.

Ovulation and the corpus luteum By the end of the follicular (or proliferative) phase of the thirteenth day of the menstrual cycle, the layer of the preovulatory follicle will develop an opening, or stigma, and excrete the oocyte with a complement of cumulus cells in a process called. In natural cycles, ovulation may occur in follicles that are at least 14 mm.The oocyte is technically still a secondary oocyte, suspended in the metaphase II of meiosis.

It will develop into an ootid, and rapidly thereafter into an ovum (via completion of meiosis II) only upon fertilization. The oocyte will now travel down one of the to eventually be discharged through menstruation in the case that it is unfertilized or if it is not successfully implanted in the (if previously ).The ruptured follicle will undergo a dramatic transformation into the, a steroidiogenic cluster of cells that maintains the of the uterus by the secretion of large amounts of and minor amounts of.These two steps, while not part of folliculogenesis, are included for completeness. They are discussed in their entirety by their respective articles, and placed into perspective by the article.

It is recommended that these three topics be reviewed.Hormone function As with most things related to the reproductive system, folliculogenesis is controlled by the. Five hormones participate in an intricate process of positive and negative feedback to regulate folliculogenesis. They are:. (GnRH) secreted by the. two:.

(FSH). (LH).GnRH stimulates the release of FSH and LH from the anterior pituitary gland that will later have a stimulatory effect on follicle growth (not immediately, however, because only antral follicles are dependent on FSH and LH). When theca cells form in the tertiary follicle the amount of estrogen increases sharply (theca-derived androgen is aromatized into estrogen by the granulosa cells).At low concentration, estrogen inhibits gonadotropins, but high concentration of estrogen stimulates them. In addition, as more estrogen is secreted, more LH receptors are made by the theca cells, inciting theca cells to create more androgen that will become estrogen downstream.

This positive feedback loop causes LH to spike sharply, and it is this spike that causes ovulation.Following ovulation, LH stimulates the formation of the corpus luteum. Estrogen has since dropped to negative stimulatory levels after ovulation and therefore serves to maintain the concentration of FSH and LH.

Inhibin, which is also secreted by the corpus luteum, contributes to FSH inhibition.The endocrine system coincides with the menstrual cycle and goes through thirteen cycles (and thus thirteen LH spikes) during the course of normal folliculogenesis. However, coordinated enzyme signalling and the time-specific expression of hormonal receptors ensures that follicle growth does not become disregulated during these premature spikes.Number of follicles. ' Percentage of ovarian reserve related to increasing age. The curve describes the percentage of ovarian reserve remaining at ages from birth to 55 years, based on the ADC model. 100% is taken to be the maximum ovarian reserve, occurring at 18–22 weeks post-conception. The percentages apply to all women whose ovarian reserve declines in line with our model (i.e. Late and early menopause are associated with high and low peak NGF populations, respectively).

We estimate that for 95% of women by the age of 30 years only 12% of their maximum pre-birth NGF population is present and by the age of 40 years only 3% remains.:'Recently, two publications have challenged the idea that a finite number of follicles are set around the time of birth. Renewal of ovarian follicles from germline stem cells (originating from bone marrow and peripheral blood) was reported in the postnatal mouse ovary. Studies attempting to replicate these results are underway, but a study of populations in 325 human ovaries found no supporting evidence for follicular replenishment.In 2010, researchers at the determined that by the time women are 30 years old, only 10% of their non-growing follicles (NGFs) remain. At birth, women have all their follicles for folliculogenesis, and they steadily decline until.Depletion of the ovarian reserve As women (and mice) age, double-strand breaks accumulate in their primordial follicle reserve.

These follicles contain primary oocytes that are arrested in prophase of the first cell division of meiosis. Double-strand breaks are accurately repaired during meiosis by searching for, and building off of, the matching strand (termed “homologous recombinational repair”). (2013) found that, as humans (and mice) age, expression of four key DNA repair genes necessary for homologous recombinational repair declines in oocytes. They hypothesized that DNA double-strand break repair is vital for the maintenance of oocyte reserve, and that a decline in efficiency of repair with age plays a key role in the depletion of the ovarian reserve (ovarian aging).See also. ^, section 'formation of the antrum' in: Sherwood, Lauralee. Human physiology: from cells to system.

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'In-vivo and in-vitro development of preantral follicles'. 47: 73–82. ^ Wallace, WHB; Kelsey, TW (2010). 5 (1): e8772. Fortune J, Cushman R, Wahl C, Kito S (2000). 'The primordial to primary follicle transition'.

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5, page 508. ^ Baerwald, Angela R.; Walker, Randy A.; Pierson, Roger A. 'Growth rates of ovarian follicles during natural menstrual cycles, oral contraception cycles, and ovarian stimulation cycles'. Fertility and Sterility.

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Kelsey (2010-01-27). 5 (1): e8772. Johnson J, Bagley J, Skaznik-Wikiel M, Lee H, Adams G, Niikura Y, Tschudy K, Tilly J, Cortes M, Forkert R, Spitzer T, Iacomini J, Scadden D, Tilly J (2005).

'Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood'. 122 (2): 303–15. Johnson J, Canning J, Kaneko T, Pru J, Tilly J (2004). 'Germline stem cells and follicular renewal in the postnatal mammalian ovary'.

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Reprod Biomed Online. 10 (6): 774–85. Fortune, JE; Yang, MY; Muruvi, W (2010). Soc Reprod Fertil Suppl. 67: 203–16. Gougeon A (1996). 'Regulation of ovarian follicular development in primates: facts and hypotheses'.

17 (2): 121–55. Gougeon A (1986). 'Dynamics of follicular growth in the human: a model from preliminary results'. 1 (2): 81–7. van den Hurk R, Zhao J (2005).

'Formation of mammalian oocytes and their growth, differentiation and maturation within ovarian follicles'. 63 (6): 1717–51. Uzumcu, Mehmet; Zachow, Rob (2007). Reproductive Toxicology. 23 (3): 337–352.External links.

Open Access is an initiative that aims to make scientific research freely available to all. To date our community has made over 100 million downloads. It’s based on principles of collaboration, unobstructed discovery, and, most importantly, scientific progression. As PhD students, we found it difficult to access the research we needed, so we decided to create a new Open Access publisher that levels the playing field for scientists across the world. By making research easy to access, and puts the academic needs of the researchers before the business interests of publishers. In many mammalian species, including humans, folliculogenesis begins in fetal life and progresses throughout adulthood. The growing follicles progress from a reserve of primordial follicles that constitute the pool of female gametes for the entire life.

Primordial follicles may begin to grow either immediately after forming or at clearly defined species-specific gap. Alternatively, some follicles may become quiescent before they either degenerate or resume growth several months or years afterwards. The rate of follicular assembly and the primordial to primary follicle transition is a critical step in female fertility. Therefore, disturbed coordination of the formation of primordial follicles and activation of their growth may entail some reproductive disorders. A poor initial reserve or the precocious primordial follicle depletion will result in infertility that, in women, is escorted by a shortened reproductive lifespan and early menopause. Therefore, it seems necessary to reach a profounder understanding of the molecular and cellular mechanisms controlling follicular development during preantral transition.

In vitro growth of isolated immature ovarian follicles (IVGF) appears as an emerging technology, allowing to expand the fertility options in particular ovarian disorders or after cancer treatment. In the ovary of a mammalian female, the process of folliculogenesis begins during fetal life and proceeds until the end of reproductive capacity, which is manifested in cell proliferation and differentiation ,. Folliculogenesis, involving growth and development of ovarian follicles from primordial to preovulatory stages, is a complex phenomenon requiring multidirectional regulation. The ovarian follicle plays an essential role in securing optimal conditions for oocyte maturation and its release during ovulation, for which it will provide an appropriate microenvironment based on locally produced molecules, such as sex steroids and peptide hormones, growth factors, and cytokines, while also providing the appropriate communication among particular compartments of an ovarian follicle –. Sex steroids produced by follicular cells are known to play one of the main roles in the regulation of ovarian function. These steroids present in the systemic circulation actively participate in the regulation of pituitary gonadotropin secretion. On the other side, sex steroids present in the ovarian microenvironment act as paracrine factors important for the maintenance of follicular development.

The majority of information about the role of sex steroids in ovarian functioning has been obtained in studies directed at the action of estrogens and progestagens. Nowadays, increasing attention is being devoted to the action of androgens because the activation of androgen receptors (ARs) located in follicular cells , modulates the expression and activity of many genes vital for the maintenance of follicular development –.From the initial pool of ovarian follicles recruited to grow, only a few reach a preovulatory stage. Less than 1% of follicles elude the process of atresia at various stages of development, and the preantral to early antral transition is the most susceptible to this process. The pool of primordial follicles established in fetal life constitutes a reserve that will not increase during the postnatal period. The initial stages of folliculogenesis, including the accumulation of primordial follicles, the recruitment of primordial follicles from the resting pool, and their transition into primary follicles, are crucial for the female reproduction regardless of the species. Improper coordination of the formation of primordial follicles and activation of their growth may entail disturbed folliculogenesis in mature individuals manifested by a reduction of fertility. Recent research has revealed that primordial and primary follicles might not die by classical apoptosis.

It is therefore possible that, in the immature ovary, other mechanisms are involved in follicular atresia.The main factor determining the selection of follicles into the antral stage is their ability to respond to gonadotropins, especially follicle-stimulating hormone (FSH). Preantral follicles display an increase in the number of FSH receptors (FSHR) that, when activated, stimulate granulosa cell proliferation, antrum formation, and biosynthesis of estradiol after the activation of aromatase enzyme. There is quite ample evidence that follicle development is dependent on their granulosa layer, the functioning of which is influenced by endocrine, paracrine, and autocrine mechanisms. Granulosa cells are involved in the control of oocyte maturation and proper execution of ovulation and participate in early embryogenesis, maintenance of corpus luteum function, and production of chemotactic factors and those involved in angiogenesis.

Sustained oocyte growth depends on the effective communication and crosstalk between granulosa cells and the oocyte, because granulosa cells remain the major source of nutrients for the gamete through homologous and heterologous gap junctional contacts.The tool that allows studying the function of ovarian follicles irrespective of its complicated structure is the model of whole organ culture, which reflects the conditions and complicated interactions occurring in vivo. These kinds of cultures constitute very sensitive objects to test the biological activity of various factors; they allow to observe the responses to increased or decreased steroid hormone secretion, the induction or inhibition of cell proliferation, or the induction or inhibition of apoptosis.

The technological revolution in reproductive biology that started with artificial insemination and embryo transfer technologies during the last 30 years has continued with oocyte in vitro maturation (IVM), in vitro fertilization (IVF), or in vitro embryo culture (IVC), to name only a few. IVM has particular significance, providing the technology platform for the abundant supply of mature, good quality oocytes for diverse applications, such as reducing the generation interval in important species or studying in vitro human reproduction. Despite the convenience of IVM, we still do not understand the precise factors and conditions occurring in vivo, which yield the highest-quality mature oocytes for successful fertilization and embryo development outcomes; hence, we cannot completely imitate these conditions. Thus, in vitro growth of isolated immature ovarian follicles (IVGF) appears as an emerging technology allowing to expand the fertility options, particularly in young cancer patients –, and may serve as a potential source of fertilizable gametes. Thus, assisted reproductive technologies allied to a profound understanding of granulosa/oocyte interactions can benefit from the capability to sustain primordial and primary follicle growth in vitro while supporting the acquisition of oocyte competence.On this basis, the objective of this chapter is to review relevant data concerning the molecular factors crucial to the regulation of early stages of folliculogenesis and to provide basic information to the design of future culture strategies promoting the in vitro development of ovarian follicles. In the mammalian embryo, ovarian development begins between 3 and 6 weeks after conception. During this period, ovarian rudiment is massively colonized by mesonephric cells, which are regarded as the follicular cell precursors, and the primordial germ cells (PGC) migrate into the genital ridge; hence, other events take place, such as the differentiation of the gonads according to gender, proliferation, and apoptosis ,.

Oocyte development begins in the mammalian female fetus together with the differentiation of PGC. Proliferating PGC migrate towards the nascent genital ridges, where they differentiate into oogonia, before entering the first meiotic division to become primary oocytes.Mammalian oocytes develop and reach ovulatory maturity inside the follicles where they are covered at first by pre-granulosa and then by granulosa cells. Over the lengthy process of follicle development, granulosa cells proliferate and the theca layer is formed , allowing the follicle to take advantage of blood supply. Then, follicles pass through the succeeding stages of development before reaching full maturation and the ability to ovulate. Primary oocytes, which are arrested at diplotene of the first meiotic prophase since late prenatal life in most mammal species, are the organizing centers of primordial follicles. The oocyte is considered to play the most important role in follicular organization during folliculogenesis. It is assumed that the oocyte controls both the proliferation and the differentiation of granulosa cells into cells capable of secreting steroids and various proteins.

On the contrary, several oocyte features, such as growth, differentiation, meiosis, cytoplasmic maturation, or control of transcriptional activity, are dependent on the presence and contact with granulosa cells. Interestingly, when the oocyte reaches a certain size threshold, it secretes factors that inhibit the ability of granulosa cells to promote its own growth , which suggests that the oocyte may determine not only its own growth but also the growth of the whole follicle. Figure 2.Mechanisms of androgen actions in follicular development. Physiological functions of androgens during primordial follicle recruitment are mediated through androgen response element (ARE)-dependent genomic actions and/or via PI3K/Akt nongenomic signaling pathway.GDF-9, growth differentiation factor-9; AR, androgen receptor.The idea that androgens might regulate follicular development initially started with studies indicating AR expression in the different compartments of follicles throughout most stages of folliculogenesis –.

However, AR expression pattern may differ between cell types, and in most species, AR is abundant in the preantral/antral stages of follicular development but declines as a follicle matures to the preovulatory stage –. Based on these observations, it was suggested that androgens might differentially regulate various stages of follicular development through an autocrine and/or paracrine way. It is generally accepted that androgens primarily affect preantral follicles and that their activities are important for preantral follicle growth and prevention of follicular atresia.

Moreover, it seems possible that androgens are involved in the activation of primordial follicles –. How androgens influence primordial follicle recruitment and whether this is a primary or secondary response to androgens are still open-ended questions needing further investigation.The mechanisms of primordial follicle activation can be studied using in vitro culture methods. However, until now, the success of primordial follicles culture as a method of oocyte growth has been limited to mice.

Eppig and O’Brien were the first to obtain mouse offspring derived from oocytes acquired from cultured primordial follicles. As to other species, several studies carried out in farm animals and primates showed that the transition of primordial into primary follicles in culture of cortical strips from caprine , bovine , baboon , and human ovaries is possible.

A confirmation of the normality of follicle development in vitro was obtained through the changes in follicle morphology and cell number as well as from the stage-specific follicular responsiveness to above-mentioned factors or the development of steroidogenic capacity. In vitro follicle growth is a promising fertility preservation strategy , despite that, in some mammalian species, including humans and pigs, the success has been limited when the process started with primordial follicles. This could be explained by the fact that adequate isolation methods and culture strategies have not yet been fully established, thereby impairing the ability to obtain mature gametes from the culture of isolated primordial follicles in those species. The manipulation of primordial follicles is a challenge due to their small size and the existing physical connections between the oocyte and the surrounding squamous granulosa cells, which are also poorly studied. Conversely, the conditions that support their activation and growth are not well defined.

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Several studies have indicated that primordial and primary follicles rapidly degenerate in cultures carried under multiple conditions –. For example, primordial follicles isolated from human ovarian tissue using collagenase digestion and subsequently cultured in collagen gels resulted in the degeneration of the follicles within 24 hours.The species and the reproductive age of the ovarian tissue affect preantral follicle yield in the ovary because of the existence of a larger number of follicles and the easiness of the isolation method in neonatal and prepubertal ovaries compared with mature ovaries ,. The success of either culture or transplantation of isolated follicles depends on the high quality of retrieved follicles. That is why an effective method for retrieving viable, preantral follicles is an essential condition.

Different methods are currently available to isolate follicles for preantral follicle culture. The mechanical isolation methods include the use of fine-gauge needles or forceps to isolate follicles from mice , rats , pigs , cattle , and humans ; the combination of ovarian dissociation methods, such as grating or mincing, with sieving ; and the follicular dissection from the ovarian cortex using a skin-grafting knife and/or small scalpel blades ,. The mechanical isolation methods have a main advantage, as they allow retrieving intact follicles, surrounded by the basement membrane and theca layers, although they are slow and laborious techniques that typically yield only a small number of follicles. These technical problems can be avoided by the use of enzymes to aid follicle recovery. The incubation of ovarian tissue in collagenase and/or DNAse (e.g. ,) softens and disaggregates the tissue matrix and allow detaching follicles from the surrounding stroma with the aid of needles. However, the degradation of the basement membrane and the absence of theca cell layers are the most common undesirable consequences of the use of enzymes in follicle isolation , as they foster the spontaneous loss of granulosa cells from the follicles in culture.

Nevertheless, the time of enzyme exposure can be controlled to minimize the damage.The ovarian stroma is dense and fibrous; thereby, it is more efficiently isolated using a combination of mechanical and enzymatic procedures that have been shown to preserve follicle viability ,–. Dolmans et al.

developed a new isolation protocol using Liberase Blendzyme 3. This blend of purified enzymes allowed the isolation of a high number of preantral follicles, which were viable as well as morphologically and ultrastructurally normal. However, this type of Liberase is no longer produced. Therefore, the second generation of Liberase DH (Dispase High) Research Grade has been successfully tested for the preparation of human ovarian follicles. However, the efficiency of the mixture may vary with the species.

Our group recommends the use of Liberase TH (Thermolysin High) Research Grade to obtain a high number of fully isolated primordial follicles from porcine ovarian cortex , as it presents a really fibrous tissue. Using prepubertal gilt ovaries, we applied different types of Liberase (DH, TM, and TH) Research Grade and treatment protocols to isolate primordial follicles. The quality of the isolated follicles was evaluated by their general morphology and viability upon routine hematoxylin and eosin (H&E) and fluorescent staining, whereas their ultrastructure was assessed by electron microscopy. Additionally, to determine the purity of isolated follicles, a germ cell-specific protein, MSY2, was used to recognize oocytes. Liberase TH Research Grade was the mixture presenting a very high proportion of retrieved viable follicles whose majority exhibited good morphology with a complete granulosa cell layer. In addition, primordial follicles stained with either Hoechst 33342 or H&E indicated that Liberase TH Research Grade only occasionally induced atresia.

This was supported by ultrastructural studies revealing that the oolemma-follicular cell interface was well preserved, which would allow the complex to express the correct metabolic profile. The results obtained in those experiments also showed that almost all of the Liberase TH Research Grade-isolated primordial follicles were MSY2 positive. As shown in the literature , primordial and primary follicles may rapidly degenerate after isolation because of the loss of critical connections between the oocyte and the granulosa cells. It seems that Liberase is a promising alternative to collagenase treatment, allowing the use of isolated primordial follicles for further reproductive studies.

Figure 4.Morphology and ultrastructure of primordial and primary pig follicles isolated from ovarian medulla using Liberase TH-Research Grade. (A) Morphology of Liberase TH-treated pre-antral follicles (light microscopy), (B) morphology of Liberase TH-isolated pre-antral follicles stained with hematoxylin and eosin; (b) interrupted granulosa cells layer in pre-antral pig follicles isolated with collagenase (type II); (C) Transmission electron microscopy (TEM) showed a single uninterrupted layer of cuboid follicular cells (black asterisk) surrounding the oocyte (white asterisk), which was bordered by a continuous basal lamina (arrow). Scanning electron microscopy (SEM) of primordial (D) and primary (E) follicles isolated using Liberase TH. A continuous layer of cuboid follicular cells surrounds the oocyte in the primary follicle (E) while in the primordial follicle a flattened layer of cells covers the oocyte; immunoconfocal images recorded from three selected areas of centrifuged ovarian digest: follicles were stained for actin (F), MS2Y (G) and with DAPI (H), merged images (I). The clinical application of IVFG is still at the investigational stage, in a laboratory setting, although it stands a robust approach to study the basic biology of the ovary or the follicle under a controlled yet adjustable environment.

Multiple culture systems have been developed to support the development of isolated preantral follicles , each one with its own advantages and providing useful insights into the follicle physiology. By this time, hydrogel-based follicle culture systems have been well characterized. The oocyte and the surrounding granulosa cells interact with each other and the environment, maintaining the same spatial location, connections, and dimensionality as in the intact ovary. The in vitro growth and development of mouse preantral follicles was successfully supported by alginate-based hydrogels, a substrate that was also applied to several large mammalian species, including dogs , rhesus monkeys , and humans , resulting in stage IV oocytes (human) , meiosis II (MII)-arrested eggs, and fertilized two-cell embryos (rhesus macaque). This developmental stage has not been reached in other systems.It is commonly agreed that early follicular growth is largely independent of a gonadotropin stimulus; instead, it seems that it is controlled by paracrine and autocrine signals originating from several sources in the ovary, including stromal cells, macrophages, and other follicles.

Recent studies showed that these local factors may also play an important role in in vitro culture, supporting the growth of isolated preantral follicles: isolated primary ovarian follicles survived and grew when cocultured with purified ovarian stroma including theca-interstitial cells and macrophages or with mouse embryonic fibroblasts (MEFs) as a feeder cell layer. Coculture with MEFs resulted in an increased follicle survival, growth, and differentiation until antral follicles contained meiotically competent oocytes capable of reaching metaphase II in response to adequate hormone stimulation. In summary, the ability to sustain preantral follicle growth in vitro while supporting the acquisition of oocyte competence is of great scientific interest. This relies on supplying oocytes for assisted reproductive technologies and broadening our understanding of somatic cell/oocyte interactions in species characterizing by prolonged follicular growth, such as humans and pigs.

IVGF is becoming a useful tool to assess follicular development, offering also the potential to preserve reproductive options in cases of polycystic ovarian syndrome (PCOS), premature ovarian failure, or definitive sterility (post-oncotherapy). In addition, it is known that certain ovarian dysfunctions, such as PCOS and gonadotropin poor responsiveness, are consequences of deregulated follicle growth at this transitional stage. Therefore, the elucidation of molecular and cellular mechanisms involved in the control of follicular development during transition from preantral to early antral stage may provide an important insight into the pathophysiology and rational treatment of these disorders. Duda Malgorzata, Grzesiak Malgorzata, Knet-Seweryn Malgorzata and Zbigniew Tabarowski (March 23rd 2016). The Primordial to Primary Follicle Transition — A Reliable Marker of Ovarian Function, Insights from Animal Reproduction, Rita Payan Carreira, IntechOpen, DOI: 10.5772/62138. Available from: Duda Malgorzata, Grzesiak Malgorzata, Knet-Seweryn Malgorzataand Zbigniew Tabarowski (March 23rd 2016). The Primordial to Primary Follicle Transition — A Reliable Marker of Ovarian Function, Insights from Animal Reproduction, Rita Payan Carreira, IntechOpen, DOI: 10.5772/62138.

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