There is no neo-oogenesis in the adult mammalian ovary

Neo-oogenesis: Has its existence been proven?

Energy status and ovarian follicular development

Immunohistochemical localization of the Fas antigen in the infant and adult human ovary during follicular growth, regression, and atresia was examined by the avidin/biotin immunoperoxidase method with a monoclonal antibody to the Fas antigen. Western blotting was used to confirm the presence of the Fas antigen protein. In primordial and primary follicles within the normal adult ovary, only the oocyte showed moderate immunostaining for the Fas antigen. In secondary and antral follicles, only the oocyte showed weak staining for the Fas antigen, while in preovulatory follicles, neither the oocyte nor the granulosa and theca cells were immunostained for the Fas antigen. In corpora lutea, the Fas antigen staining became apparent in the granulosa lutein cells during the early luteal phase and intensified during the mid luteal phase, while the theca lutein cells became positive for the Fas antigen staining during the mid luteal phase. During the late luteal phase, the staining intensity of the Fas antigen in the regressing corpora lutea further increased. As the regressing corpora lutea were converted into corpora albicans, the staining intensity decreased, and the corpora albicans and stromal cells were negative for the Fas antigen. In atretic primordial and primary follicles, only the degenerating oocyte showed the Fas antigen staining. By contrast, in atretic antral follicles, the Fas antigen staining was profound in the degenerating granulosa cells at the early stage of atresia, and at the mid stage of atresia it was intensified in the cell surface of the scattered granulosa cells and became apparent in the theca cells. At the late stage of atresia the Fas antigen remained only in the hypertrophied theca cells. In the infant ovary, only the oocyte in primordial and primary follicles exhibited intense staining for the Fas antigen. In the postmenopausal ovary, the Fas antigen staining was entirely negative. Western blot analysis revealed the presence of the Fas antigen protein with a molecular mass of 45 kDa in luteal tissues. On the basis of the recent evidence, that the Fas antigen mediates an apoptotic signal in a variety of cells, the abundant expression of the Fas antigen in the regressing corpora lutea and atretic follicles suggests that the Fas antigen participates in luteal regression and follicular atresia through the apoptotic process. Furthermore, notable expression of the Fas antigen in the oocyte of primordial and primary follicles within the infant and adult human ovary followed by the decrease in the Fas antigen expression in the oocyte with the advance of follicular maturation suggests that the Fas antigen expression in the oocyte may play a role in follicular selection.

Not yet available.

Up to now, the accepted wisdom was that, unlike males who retain germline stem cells (GSCs) through their adult lives, oocyte production in females of most mammalian species ceases before birth. Females therefore are born with a fixed pool of GSCs, or oocytes, all of them arrested in meiosis. The ovarian follicles are oocytes enclosed by somatic cells. The oocyte reserve in human females is exhausted at approximately the fifth decade of life, inducing menopause. Directly contradicting these views, the investigators have found that juvenile and adult mouse ovaries have mitotically active germ cells that continuously replenish the pool of follicles. A study of mouse ovaries, based on analyses of healthy quiescent and early growing follicle numbers, showed that approximately one third of the peak endowment of immature follicles was lost by young adulthood. In contrast to previous studies in mammals, a parallel study of atretic (degenerating) follicles showed that atresia increased markedly by days 30 to 40, reaching a peak of more than 1200 dying follicles per ovary that continued well into reproductive life (Fig. 1). There was a clear discordance between changes in numbers of nonatretic follicles and the corresponding incidence of atresia in the postnatal mammalian ovary. Histologic studies demonstrated large ovoid cells that resembled germ cells of the fetal mouse ovary; they were found in the surface epithelial cell layer covering the ovary. Immunohistochemical studies affirmed that these cells were of a germline lineage. Immunostaining confirmed the presence of germ cells in varying stages of meiosis. The investigators identified genes whose expression is necessary for replicating germ cells to produce oocytes for follicle formation in postnatal life. In studies with busulphan, a germ-cell toxicant, exposed ovaries had fewer than 5% of the primordial follicle pool present in control animals. The rate of follicle loss was very close to that expected from previously reported data. Finally, ovarian fragments taken from wild-type mice were grafted into the ovaries of transgenic female siblings after removing approximately half of the host’s ovary. The transgenic germ cells infiltrated the grafted tissue and initiated folliculogenesis with the resident wild-type somatic cells. These observations suggest that GSCs are present in the postnatal mouse ovary. The findings have therapeutic potential for expanding the follicle reserve so as to postpone normal or premature ovarian failure.Fig. 1: Postnatal ovarian germ-cell dynamics. (A and B) Numbers of nonatretic (A) and atretic (B) primordial and total immature (primordial, primary, small preantral) follicles in mouse ovaries during postnatal development (B, total immature follicles; mean ± standard error, n = 3–4 mice per age group). (C) Incidence of primordial and primary follicle atresia in ovaries exposed to DMBA on day 25 postpartum (mean ± standard error, n = 4–5 mice per time point). (D) Comparison of nonatretic and atretic immature follicle numbers in C57BL/6, CD1, and AKR/J strains of mice (mean ± standard error, n = 3–5 mice per age group). Reproduced with permission. Nature 2004;428:145–150. Copyright © 2004, Nature Publishing Group. All rights reserved.

Study question How does the ovarian microenvironment, involving neural fibers, vascular networks, and immune cells, interplay with follicular dynamics in the human adult ovary? Summary answer Neural fibers growing into the ovarian cortex and vascular network expansion were associated with primordial follicle activation and growth. Macrophages associated with antral follicular atresia. What is known already The ovarian follicles, responsible for preserving and supplying oocytes, play a crucial role in fertility, with their fate influenced by the ovarian microenvironment. However, the three-dimensional (3D), irregular, and dynamic structural changes of the ovarian microenvironment are difficult to interpret using two-dimensional analysis. 3D-imaging technologies have been developed to reveal intricate structural and functional relationships particularly in rodents, but these approaches have not been widely adopted for human ovarian research. Study design, size, duration Our study aims to explore the follicular microenvironment dynamics in the human adult ovary. In this work, we optimized whole-mount immunofluorescence using TUBB3, PODXL, PECAM1, ACTA2 and CD68, and conducted 3D imaging on intact adult ovarian tissue, offering a visualization of the 3D structures and dynamic changes within ovarian follicles, neural fibers, vascular networks, and immune cells in human adult ovary at the single-cell level. Participants/materials, setting, methods The human ovarian samples were collected from donors (N = 5) who underwent oophorectomy after testosterone treatment. Part of the intact ovary, including cortex and medulla, was fixed and used for whole-mount immunofluorescence using markers for neural fibers (TUBB3), blood vessels (PODXL, PECAM1, ACTA2), and immune cells (CD68). Subsequently, an enhanced iDiSCO method was employed for tissue clearance. Finally, 3D images were acquired using a lightsheet microscope, followed by image analysis and 3D reconstruction using Imaris software. Main results and the role of chance Our results showed that TUBB3-labeled nerve fibers predominantly distributed in the ovarian cortex, with few presented in the medullary region. Notably, their presence surrounding secondary and antral follicles was limited. We observed a rich distribution of PODXL-labeled capillaries surrounding primordial and primary follicles in the cortical layer of the adult ovary. As follicles progressed to the secondary stage, PECAM1-labeled vasculature contributed to the construction of a vascular network enveloping the secondary follicles and connecting extensively to larger-caliber arteries containing (ACTA2+) smooth muscle cells. In antral follicles, large larger-caliber arteries were also detected, maintaining proximity to the theca cell layer. Interestingly, in cases of follicular atresia, vascular invasion within the follicular cavity was observed. Additionally, clusters of CD68+ macrophages exhibited a scattered distribution around the follicles at various stages, with a significant accumulation in atretic small-antral follicles. Limitations, reasons for caution Our study was conducted on ovaries from a limited number of donors after gender-affirming surgery. Although we aimed to provide insights into the ovarian microenvironment, we were so far unable to obtain ovaries with large antral follicles, ovulatory follicles and the formation of the corpus luteum. Wider implications of the findings The views provided by 3D-image analysis of the human ovary will augment the current knowledge of the complex interactions between the follicles and the ovarian microenvironment. 3D ovarian mapping will also provide a roadmap for generating oocytes in vitro for fertility preservation and ovarian disease modelling. Trial registration number N/A.

The glycoside residues (glycoconjugates, GC) of the zona pellucida (ZP) glycoproteins are important during the first phases of fecundation. Our aim in this work was to determine the lectin affinity pattern of porcine ZP in order to analyze the changes that take place during: (a) preantral folliculogenesis, (b) the follicular atresia process, and (c) antral growth. Several prepubertal and adult pig ovaries and different sized antral follicles were used. Conventional carbohydrate histochemical techniques and peroxidase and digoxigenin (DIG) lectins were used to reveal the acid groups and the glycosidic residues of the ZP. It was seen that the ZP forms in the preantral follicles throughout their growth period. In primordial and primary follicles, ZP in the process of formation showed neutral GC. SBA, RCA-I, MAA, WGA lectins, and AAA after methylation-saponification (MS) were positive in the ZP of primordial and primary follicles. The affinity for SBA, RCA-I, MAA, and WGA increased in the multilaminar-primary follicles and new affinities for UEA-I and LFA were observed. After MS, AAA, SNA, PNA, and SBA reactivity was observed. The ZP of antral follicle oocytes of different sizes showed the same lectin pattern as multilaminar-primary follicles. The oocyte ooplasm and the follicular fluid of large antral follicles showed less affinity for WGA and LFA lectins and less intensive staining with AB (pH 2.5). Atresia did not change the antral or preantral follicle oocyte ZP lectin pattern. In conclusion, the follicles showed substantial changes in their ZP glycosidic composition as they developed, especially, during the change from primary to multilaminar-primary follicles. The ZP glycosidic composition showed no significant change during the growth of antral follicles and follicular atresia in our study.

The expression patterns of pro-apoptotic and anti-apoptotic factors in human fetal and adult ovary

The aim of the present study was to investigate the sites and time of follistatin and inhibin alpha and beta A subunit gene expression during ovine follicular development and atresia. Prepubertal ovaries of 2-, 8- and 14-week-old ewe lambs (n = 9) were used. Regardless of age, the ovaries contained many follicles at different stages of development up to 2 mm in diameter, but large antral follicles were not found. Ovarian sections were hybridized with 35S-labelled antisense RNA probes transcribed from follistatin, inhibin alpha or inhibin beta A cDNA. Ovaries from mature gonadotrophin-stimulated ewes were used as controls. All three probes hybridized exclusively to granulosa cells, and not to other follicular or stromal cells. None of the probes hybridized to primordial follicles or primary follicles with less than two layers of granulosa cells. Follistatin mRNA was expressed strongly in the granulosa cells of all preantral follicles with two or more layers of cells, and in all non-atretic antral follicles. In addition, follistatin mRNA was found in some cells of the ovarian rete tubules. The inhibin alpha riboprobe hybridized to the granulosa cells of most preantral and all non-atretic antral follicles. In the preantral follicles, the strongest inhibin alpha expression was observed in the cells that were in close proximity to the oocyte. The inhibin beta A riboprobe hybridized exclusively to the granulosa cells of antral follicles. The labelling was observed either in the cumulus oophorus or in the cumulus oophorus and periantral granulosa cells of the non-atretic antral follicles. In adult ovaries, which were used as controls, the inhibin beta A riboprobe hybridized strongly to all granulosa cells of non-atretic large antral follicles. During follicular atresia, expression of all three mRNAs progressively decreased. In early atresia, inhibin beta A mRNA was observed only in the cells of the cumulus, whereas inhibin alpha and follistatin mRNAs were still present in the granulosa cells. As atresia progressed mRNA for inhibin alpha, and later also follistatin, disappeared. Our results suggest that there is a sequential appearance and disappearance of follistatin, inhibin alpha and inhibin beta A mRNAs during follicular development and atresia respectively. The marked expression of inhibin beta A in the cumulus cells implies a role of the oocyte in the differentiation of these cells.

The human ovary is a complex endocrine gland, which is responsible for production of different hormones and provides mature and competent oocytes for reproduction. Additionally, it produces various substances, such as growth factors and cytokines which are involved in the complex signalling pathways of folliculogenesis or oogenesis. The abnormalities of ovarian function might lead to infertility or manifestation of aggressive cancer. Therefore, stem cells in adult human ovaries are of great interest to reproductive medicine for improved understanding of the mechanisms leading to ovarian infertility or cancer formation, yet they represent a difficult scientific subject, because it is still generally accepted that they do not exist. The persisting dogma is that the end number of follicles is set up at the time of birth, and that there is no neo-folliculogenesis or neo-oogenesis in the postnatal or adult ovaries. The main reason for persistence of the dogma lies in the fact that it is very difficult to perform studies on adult human ovaries; it is impossible to perform in vivo studies, and there is also a lack of ovarian tissue available for research. However, there is more and more evidence about the presence of putative stem cells in postnatal and adult mammalian ovaries. First promising experimental results were obtained in the mouse model, but have been followed also in humans. The aim of this review article is to elucidate the fast upcoming new knowledge of ovarian stem cells, and their potential implications for reproductive medicine and gynecological oncology in the future.

Ovarian follicular development is dependent on growth and differentiation of the oocyte, as well as the granulosa and theca cell layers. The majority of primary follicles in the adult human ovary are not growing, and most antral follicles undergo atresia. The mechanisms regulating follicular growth and differentiation are poorly understood. Expression of key regulatory proteins in cells of certain follicles may be involved. We have studied the distribution of retinoblastoma protein (pRb), a key cell cycle regulator, in human follicles and CL by quantitative immunohistochemistry. Recent studies suggest that high nuclear concentrations of pRb are associated with the arrest of cell proliferation and the beginning of differentiation; during advanced differentiation of cells pRb is markedly depleted or absent. We also studied follicular distribution of Thy-1 differentiation protein, a morpho-regulatory molecule associated with cell differentiation, and the presence of macrophages. Macrophages have been shown to stimulate steroidogenesis in granulosa cells in vitro, and they are required for release of Thy-1 differentiation protein from vascular pericytes among granulosa cells in vivo. Our results indicate that oocytes in resting follicles exhibit pRb in the nucleoli. During initiation of follicular growth, the pRb expression first extends over the oocyte nuclei and then diminishes from both nuclei and nucleoli in preantral follicles. When the oocytes reach maximum size in small antral follicles, the pRb expression is reestablished in oocyte nucleoli. In differentiating granulosa and theca cell layers of preantral and small antral follicles, pRb expression is high, but it is low in growing large antral follicles. During CL development and regression, pRb expression in the nuclei of granulosa lutein cells first increases and then decreases. Follicular development is accompanied by the presence of Thy-1 differentiation protein and macrophages under the follicular basement membrane. In growing large antral follicles, during the mid-follicular phase, larger macrophages exhibit physical contacts with granulosa cells through the follicular basement membrane, and, during the late follicular phase, small dendritic macrophages can be detected among granulosa cells, but not within the follicular antrum. Large antral follicles undergoing atresia exhibit strong pRb expression in granulosa cells. This is accompanied by a lack of Thy-1 differentiation protein among granulosa cells and the occurrence of large phagocytic macrophages in the follicular antrum. This is the first report of pRb expression in the human ovary.(ABSTRACT TRUNCATED AT 400 WORDS)

The cytologic localization and cellular levels of tumor necrosis factor-alpha (TNF alpha) in the human ovary during follicular growth and regression were examined by the avidin/biotin immunoperoxidase method with a specific antibody to human recombinant TNF alpha. In the infant ovary, TNF alpha immunostaining was present only in the oocyte within the primordial follicles. TNF alpha immunostaining was also present within the oocyte in primordial follicles of the adult ovary. Positive immunostaining for TNF alpha in granulosa cells became apparent in preantral follicles, while that in theca interna cells began to appear at the antral follicle stage. The staining intensity of the oocyte increased as the oocyte reached the preovulatory stage. The intensity of immunostaining for TNF alpha in the granulosa and thecal cells increased as the follicle became larger and matured. In corpora lutea, the immunostaining for TNF alpha persisted in the granulosa lutein and theca lutein cells and intensified in the mid luteal phase. In the regressing corpora lutea, TNF alpha immunostaining in the luteal cells decreased as the regression advanced. Regressed corpora lutea with a central core of scar tissue were bordered by macrophage-like cells which exhibited intense immunostaining for TNF alpha. In the cortex region, the corpus albicans was negative for TNF alpha immunostaining, whereas macrophage-like cells peripheral to the corpus albicans exhibited intense immunostaining for TNF alpha. In the medullary region, the corpus albicans and surrounding stromal cells were totally negative for TNF alpha. By contrast, in the early atretic stage, the degenerating oocyte showed weak immunostaining for TNF alpha, while the granulosa and theca interna cells showed moderate immunostaining for TNF alpha. In the late atretic stage, the immunostaining for TNF alpha in the scattered granulosa cells became negligible, whereas the theca interna cells showed intense immunostaining for TNF alpha. The results obtained indicate that the oocyte is the primary intrafollicular site of TNF alpha localization within the ovary and that TNF alpha may participate in regulating follicular growth, regression and atresia.

The objective of this study was to analyse the expression and cellular localization of FOXO3, pFOXO3 and PTEN throughout human ovary development both before and after birth. Foetal, pubertal and adult paraffin-embedded ovarian samples were analysed by immunohistochemistry for cellular localization of FOXO3, pFOXO3 and PTEN proteins. Protein and mRNA expression were analysed by western blot and real time PCR, respectively, from fresh biopsies. PTEN was not detected by immunohistochemistry in germ cells and follicles of foetal, pubertal and adult ovaries. Occasional PTEN immunoreactive granulosa cells were found in atretic antral follicles in the adult ovary. Western blot analysis showed low levels of PTEN protein. Nuclear FOXO3-expressing primordial follicles represented a variable proportion of the ovarian reserve. The presence of FOXO3-expressing primordial follicles was very low in foetal ovary; although always represented in a low proportion, prevalence increased during pubertal and adult life. Our results seem to indicate that two subpopulations of primordial follicles, i.e. nuclear FOXO3-expressing and no FOXO3-expressing primordial follicles are found in the postnatal human ovary. This scenario suggests that FOXO3 could be acting as in the mouse model, preventing primordial follicle activation. However, the strategy would not be an "all or nothing" system as in mouse ovary but rather a selected subpopulation of primordial follicles preserved to ensure long-term fertility.

Three distinct types of gland cells occur in adult human ovaries and those of other mammals. These are: (1) Interstitial gland cells formed from the theca interna cells of degenerating (atretic) follicles, hence present from infancy to old age; (2) Thecal gland cells formed from the theca interna cells of ripening follicles, hence present only in individuals that are sexually mature or nearly so, and in these only at or near the time of ovulation; (3) Luteal cells formed from the granulosa cells of ovulated follicles and from the undifferentiated stroma cells surrounding these, and in certain species also from the same sources in relation to atretic follicles. These latter are the so‐called accessory corpora lutea. Primary and accessory corpora lutea are present normally only after ovulation occurs and appear to function for a relatively short time unless pregnancy ensues. In certain species other types of gland cells occur in the ovary and mesovarium; these include the paraluteal cells of the human ovary. Paraluteal cells are not persistent thecal gland cells, but are differentiated from surrounding stroma after the thecal gland disappears. They are probably intermediate stages in the differentiation of luteal cells from stroma cells. Planimetric measurements and computations made from serial sections of human ovaries indicate that in non‐pregnancy and early pregnancy the volume of interstitial gland tissue is less than 1% of the total ovarian volume. However, in the last trimester of gestation, the relative amount of interstitial gland tissue increases rapidly to roughly from 4% to 6% of the total ovarian volume. The largest amount in our series was 5.6%. Luteal gland tissue at the height of its development in the first trimester of gestation reached 31% of the total ovarian volume. In human ovaries the cells of the interstitial gland tissue of late pregnancy seem to be consistently larger and to have a more finely granular cytoplasm than those of early pregnancy or nonpregnancy. The literature shows that mammalian interstitial gland cells of comparable origin to the human cells discussed in this paper have the following characteristics which are known to be indicative of steroid secretors: Numerous birefringent, acetone soluble, autofluorescent granules which react with sulphuric acid and are Schultz and Schiff positive; mitochondria with anastomosing tubular cristae; and smooth tubular endoplasmic reticulum. Physiological studies so far reported have indicated possible estrogen, androgen, and progesterone production by interstitial gland cells, but all of the evidence is too scanty, indirect, and at times contradictory to be convincing. Since interstitial gland cells are present from birth to old age, and show cycles of abundance and differentiation correlated with the reproductive age and cycles, they may prove to be the most important ovarian gland cells when their function is fully understood.

Detection of connexin (Cx) proteins has been used as an indicator of the presence of structural and functional gap junctions in tissues. To examine the role of gap junctions during follicular growth and atresia, the presence of three major connexins, Cx43, Cx32, and Cx26, was evaluated in bovine ovaries by using immunohistochemistry and Western immunoblot analysis. Cx43 was not present in primordial follicles, but was present in granulosa cells of primary/secondary and antral follicles. Cx43 also was present on the borders between granulosa cells and the oocyte. Expression of Cx43 increased in healthy developing antral follicles, but decreased during follicular atresia. Cx32 was not present in healthy follicles but was present in granulosa cells of atretic antral, and especially small antral follicles. Cx26 was present in the oocyte of primordial and primary/secondary follicles, and in the granulosa and/or thecal cell layers of healthy antral follicles. The percentage of healthy antral follicles that expressed Cx26 also increased during follicular development, but decreased during atresia. Cx32 and Cx26 also were detected in ovarian blood vessels and in stromal tissues adjacent to the tunica albuginea in some ovaries. The pattern of expression of these Cx indicates that gap junctional proteins may be involved in the control of follicular growth and atresia in cows.