Oogenesis in Adult Mammals, Including Humans: A Review

Neo-oogenesis: Has its existence been proven?

There is no neo-oogenesis in the adult mammalian ovary

The central thesis regarding the human ovaries is that, although primordial germ cells in embryonal ovaries are of extraovarian origin, those generated during the fetal period and in postnatal life are derived from the ovarian surface epithelium (OSE) bipotent cells. With the assistance of immune system-related cells, secondary germ cells and primitive granulosa cells originate from OSE stem cells in the fetal and adult human gonads. Fetal primary follicles are formed during the second trimester of intrauterine life, prior to the end of immune adaptation, possibly to be recognized as self-structures and renewed later. With the onset of menarche, a periodical oocyte and follicular renewal emerges to replace aging primary follicles and ensure that fresh eggs for healthy babies are always available during the prime reproductive period. The periodical follicular renewal ceases between 35 and 40 yr of age, and the remaining primary follicles are utilized during the premenopausal period until exhausted. However, the persisting oocytes accumulate genetic alterations and may become unsuitable for ovulation and fertilization. The human OSE stem cells preserve the character of embryonic stem cells, and they may produce distinct cell types, including new eggs in vitro, particularly when derived from patients with premature ovarian failure or aging and postmenopausal ovaries. Our observations also indicate that there are substantial differences in follicular renewal between adult human and rat ovaries. As part of this chapter, we present in detail protocols utilized to analyze oogenesis in humans and to study interspecies differences when compared to the ovaries of rat females.

A year ago, reproductive biologists and general public were astonished with evidence reported by Johnson et al. in Nature 428:145 that mammalian ovaries possess persisting large germline stem cells, which allegedly enable follicular renewal in adult females. Recently, the same research group declared such view obscure, and reported that mammalian oocytes originate from putative germ cells in bone marrow and are distributed by peripheral blood to the ovaries (Cell 122:303). While neglecting available data on the germ cell origin from the ovarian surface epithelium (OSE) in adult mouse and human females and complexity of follicular renewal in humans, the authors widely extrapolated their observations on formation of allogeneic oocytes after bone marrow (or blood) transplantation in ovaries of adult mice treated with cytostatics to clinical implications in the public media. Yet, the resulting outcome that such allogeneic oocytes may enable the propagation of ovarian cycles is a poor alleviation for the women with ovarian infertility. Women lacking primary follicles, or carrying follicles with low quality eggs persisting in aging ovaries, are not concerned about the lack of menstrual cycles or ovarian steroids, but about virtually no chance of having genetically related children. Johnson et al. also reported that the germ cell formation in bone marrow disappears in ovariectomized mice. Such observation, however, raises solid doubts on the bone marrow origin of oocytes. Since germ cells developing from the OSE cells of adult human ovaries during periodical follicular renewal are known to enter blood vessels in order to enable formation of primary follicles at distant ovarian sites, they also contaminate peripheral blood and hence bone marrow. Better knowledge on the complexity of follicular renewal in humans and exploration of a potential of human OSE cells to produce new oocytes in vitro are essential for novel approaches to the autologous treatment of premature ovarian failure and age induced ovarian infertility.

In vitro maturation (IVM) and parthenogenesis of adult murine primary ovarian follicles is ameliorated in the absence of anti-mÜllerian hormone (AMH)

Not yet available.

Recent reports indicate that functional mouse oocytes and sperm can be derived in vitro from somatic cell lines. We hypothesize that in adult human ovaries, mesenchymal cells in the tunica albuginea (TA) are bipotent progenitors with a commitment for both primitive granulosa and germ cells. We investigated ovaries of twelve adult women (mean age 32.8 ± 4.1 SD, range 27–38 years) by single, double, and triple color immunohistochemistry. We show that cytokeratin (CK)+ mesenchymal cells in ovarian TA differentiate into surface epithelium (SE) cells by a mesenchymal-epithelial transition. Segments of SE directly associated with ovarian cortex are overgrown by TA, forming solid epithelial cords, which fragment into small (20 micron) epithelial nests descending into the lower ovarian cortex, before assembling with zona pellucida (ZP)+ oocytes. Germ cells can originate from SE cells which cover the TA. Small (10 micron) germ-like cells showing PS1 meiotically expressed oocyte carbohydrate protein are derived from SE cells via asymmetric division. They show nuclear MAPK immunoexpression, subsequently divide symmetrically, and enter adjacent cortical vessels. During vascular transport, the putative germ cells increase to oocyte size, and are picked-up by epithelial nests associated with the vessels. During follicle formation, extensions of granulosa cells enter the oocyte cytoplasm, forming a single paranuclear CK+ Balbiani body supplying all the mitochondria of the oocyte. In the ovarian medulla, occasional vessels show an accumulation of ZP+ oocytes (25–30 microns) or their remnants, suggesting that some oocytes degenerate. In contrast to males, adult human female gonads do not preserve germline type stem cells. This study expands our previous observations on the formation of germ cells in adult human ovaries. Differentiation of primitive granulosa and germ cells from the bipotent mesenchymal cell precursors of TA in adult human ovaries represents a most sophisticated adaptive mechanism created during the evolution of female reproduction. Our data indicate that the pool of primary follicles in adult human ovaries does not represent a static but a dynamic population of differentiating and regressing structures. An essential mission of such follicular turnover might be elimination of spontaneous or environmentally induced genetic alterations of oocytes in resting primary follicles.

The origin of oocytes and primary follicles in ovaries of adult mammalian females is still a matter of dispute [1]. The components of new primary follicles, primitive granulosa and germ cells, differentiate sequentially and de novo from mesenchymal progenitor cells residing in the ovarian tunica albuginea (TA). It appears that mesenchymal progenitor cells contribute to the generation of epithelial cells similar to granulosa cells (GCs). The multipotency of a subset of granulosa cells was also established by in vitro differentiation into other cell types [2]. Up to now, luteinizing GCs were considered to be terminally differentiated, unavoidably becoming apoptotic a few days after ovulation. Previously, we have provided evidence for the existence of putative stem cells derived from human ovarian follicular liquid collected after routine procedures for in vitro fertilization techniques [3]. These cells grow in minimal medium condition, without any growth factor (i.e. LIF), that is considered essential according to other procedures [4]. Using immunocytochemistry and flow cytometry we showed that these cells are positive for several mesenchymal stemness markers, including CD90, CD73, CD44, CD105. However, morphological analysis revealed a heterogeneous cell population, with cells displaying a fibroblast-like, epithelial- like and neural-like shapes. These observations are also supported by the identification of cells expressing specific neural markers, such as neurofilaments and PGP9.5, in addition to vimentin and cytocheratin positive cells. All these data are suggestive of the presence of different cell populations in follicular fluids. To verify this hypothesis we select a panel of markers specific for the different cell populations previously identified and we plan a molecular screening to follow their expression in the follicular fluid derived cells at different times of minimal culture conditions in vitro. Bone marrow derived MSCs were used as a control. For each sample we performed semiquantitative RT-PCR experiments normalizing the cDNAs used as templates on the basis of the number of pseudo-mesenchymal cells morphologically identified in the sample. For this purpose OCT-4 was selected as a stem marker to follow the mesenchymal stem cell population, while FSH-R was used to identify granulosa derived cells; CNTF and beta-3-tubuline were used to discriminate between neural and neuronal cells populations; epithelial and hematopoietic cells were followed using cytokeratin (CK8 and CK10) and CD45 markers, respectively. GAPDH and β-actin specific primers were used on all samples for normalization. Here we compare the results of this molecular screening with the previously obtained immunocytochemical and morphological data to confirm the presence of these different cytotypes in the samples purified from the follicular liquid and their persistence, loss or amplification at different times of in vitro minimal culture conditions.

Ten years ago, we reported that in adult human females the ovarian surface epithelium (OSE) is a source of germ cells. Recently, we also demonstrated that new primary follicles are formed by assembly of oocytes with nests of primitive granulosa cells in the ovarian cortex. The components of the new primary follicles, primitive granulosa and germ cells, differentiated sequentially from the OSE, which arises from cytokeratin positive mesenchymal progenitor cells residing in the ovarian tunica albuginea. In the present study, we investigated the possibility that the oocytes and granulosa cells may differentiate in cultures derived from adult human ovaries. Cells were scrapped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli [phenol red (PhR)]. The OSE cells cultured in the medium without PhR differentiated into small (15 micron) cells of granulosa phenotype, and epithelial, neural, and mesenchymal type cells. In contrast, OSE cells cultured in the presence of PhR differentiated directly into large (180 micron) cells of the oocyte phenotype. Such cells exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These in vitro studies confirm our in vivo observations that in adult human ovaries, the OSE is a bipotent source of oocytes and granulosa cells. Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition, other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ totipotent embryonic stem cells developing from fertilized oocytes.

Asymmetric division is a fundamental means of generating cell diversity and may involve extrinsic or intrinsic factors. Here we review observations on symmetric and asymmetric expression of estrogen receptor alpha (ERA) and beta (ERB) during regeneration of trophoblast cells in human placenta and possibly other estrogen-responsive cell types. This is a type of differentiation from committed progenitor cells. Asymmetric segregation of ERA in dividing villous cytotrophoblast cells, accompanied by appearance of ERB in differentiating daughter cells and resulting syncytiotrophoblast, suggests a unique role of estrogen receptors in asymmetric division of estrogen responsive cells. We also review observations on asymmetric division of ovarian surface epithelium (OSE) stem cells resulting in formation of germ cells differentiating into oocytes in fetal and adult human ovaries. Besides germ cells, the OSE stem cells also give rise to primitive ovarian granulosa (follicular) cells, which are required for the formation of new primary follicles and preservation and differentiation of oocytes. This dual potential of OSE stem cells (germ or granulosa cells) is a type of differentiation from uncommitted and possibly totipotent adult stem cells. A possible role of immune system related cells (monocyte-derived cells and T lymphocytes-cellular signaling) and hormones in the stimulation of OSE differentiation toward germ cells by asymmetric division, and in the continuation of ovarian follicular renewal during prime reproductive period in human females is also reviewed. Follicular renewal ceases after prime reproductive period, possibly due to the diminution of cellular signaling required for asymmetric division of OSE stem cells into the germ cells. The primary follicles persisting in premenopausal ovaries appear to accumulate genetic alterations, a cause of exponentially growing chromosomal abnormalities in the progeny of mothers between 38 years of age and menopause.

The immune system plays an important role in the regulation of tissue homeostasis ("tissue immune physiology"). Function of distinct tissues during adulthood, including the ovary, requires (1) Renewal from stem cells, (2) Preservation of tissue-specific cells in a proper differentiated state, which differs among distinct tissues, and (3) Regulation of tissue quantity. Such morphostasis can be executed by the tissue control system, consisting of immune system-related components, vascular pericytes, and autonomic innervation. Morphostasis is established epigenetically, during morphogenetic (developmental) immune adaptation, i.e., during the critical developmental period. Subsequently, the tissues are maintained in a state of differentiation reached during the adaptation by a “stop effect” of resident and self renewing monocyte-derived cells. The later normal tissue is programmed to emerge (e.g., late emergence of ovarian granulosa cells), the earlier its function ceases. Alteration of certain tissue differentiation during the critical developmental period causes persistent alteration of that tissue function, including premature ovarian failure (POF) and primary amenorrhea. In fetal and adult human ovaries the ovarian surface epithelium cells called ovarian stem cells (OSC) are bipotent stem cells for the formation of ovarian germ and granulosa cells. Recently termed oogonial stem cells are, in reality, not stem but already germ cells which have the ability to divide. Immune system-related cells and molecules accompany asymmetric division of OSC resulting in the emergence of secondary germ cells, symmetric division, and migration of secondary germ cells, formation of new granulosa cells and fetal and adult primordial follicles (follicular renewal), and selection and growth of primary/preantral, and dominant follicles. The number of selected follicles during each ovarian cycle is determined by autonomic innervation. Morphostasis is altered with advancing age, due to degenerative changes of the immune system. This causes cessation of oocyte and follicular renewal at 38 +/-2 years of age due to the lack of formation of new granulosa cells. Oocytes in primordial follicles persisting after the end of the prime reproductive period accumulate genetic alterations resulting in an exponentially growing incidence of fetal trisomies and other genetic abnormalities with advanced maternal age. The secondary germ cells also develop in the OSC cultures derived from POF and aging ovaries. In vitro conditions are free of immune mechanisms, which prevent neo-oogenesis in vivo. Such germ cells are capable of differentiating in vitro into functional oocytes. This may provide fresh oocytes and genetically related children to women lacking the ability to produce their own follicular oocytes. Further study of "immune physiology" may help us to better understand ovarian physiology and pathology, including ovarian infertility caused by POF or by a lack of ovarian follicles with functional oocytes in aging ovaries. The observations indicating involvement of immunoregulation in physiological neo-oogenesis and follicular renewal from OSC during the fetal and prime reproductive periods are reviewed as well as immune system and age-independent neo-oogenesis and oocyte maturation in OSC cultures, perimenopausal alteration of homeostasis causing disorders of many tissues, and the first OSC culture clinical trial.

A Single Dose of the Ovotoxicant 4-Vinylcyclohexene Diepoxide Is Protective in Rat Primary Ovarian Follicles

Oocyte Family Trees: Old Branches or New Stems?

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.

Can oocyte quality be augmented?