Immunoregulation of follicular renewal, selection, POF, and menopause in vivo, vs. neo-oogenesis in vitro, POF and ovarian infertility treatment, and a clinical trial

Abstract

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.

Figure 1

The prime reproductive period doctrine. The incidence of trisomic fetuses (dotted line) exponentially increases after 38+2 years of age, i.e., after the termination of follicular renewal during the prime reproductive period (PRP). White line indicates fluctuation of primordial follicle numbers due to their cyclic atresia and renewal during the prime reproductive period. PF, primordial follicles; N, neonate; CH, childhood; M, menarche; AMA, advanced maternal age. Adapted from [94] with permission, © Informa Healthcare, London, UK.

Figure 2

The human fetal ovary (24 weeks). Papanicolaou (PAP) staining and immunohistochemistry as indicated in panels. A) Secondary germ cells descend (white arrowheads) from the OSC between mesenchymal cell cords (mcc), enlarge within the cortex (black arrowhead) above the nuclear cluster (nc) or syncytium of germ cells. The arrow indicates a mesenchymal type cell. Expression of MHC class I (B), Thy-1 (C), DR of activated MDC (D), and cytokeratin (CK) accompany primordial and primary follicles (asterisks) and vessels (v). F) H&E staining of rete ovarii showing rete channels (rc). The rete shows no CK expression (G) but show high Thy-1 staining (H). The presence of CD14+ primitive MDC (I), DR+ MDC (J) and CD3 (K) and CD8 T cells (L). M) Numerous oocytes exhibit degenerative changes (vacuolization). A scale bar in (L) for panels A-L. See text for details. Adapted in part from [100] with permission, © Humana Press.

Figure 3

The human fetal ovary (24 weeks). A) Sprouts of primitive granulosa cells (pgc) originating from OSC between adjacent mesenchymal cell cords. B) In the cortex the primitive granulosa cells associate with available oocytes (asterisk). C) Pericytes (white arrow) in mesenchymal cell cords release large quantities of Thy-1 (black arrows and arrowhead) among adjacent oocytes and primitive granulosa cells. D and E) Rete cord (rc) extensions underline OSC and secrete Thy-1 (arrowheads) collapsing into spikes (arrows). F and G) Secondary germ cells (black asterisks) originating by asymmetric division of OSC (white asterisks) show depletion of MHC heavy (F) and light chain (G). Staining as indicated in panels (see Figure  2 legend). G) beta 2 microglobulin (beta 2m) = MHC class I light chain. Asterisks indicate germ cells/oocytes. Abbreviations and arrows/arrowheads are explained in the text. Adapted in part from [100] with permission, © Humana Press.

Figure 4

The human fetal ovary (24 weeks). CD14+ MDC (m, A) exhibit extensions (arrowheads) among some OSC, and accompany (B) symmetrically dividing (arrowheads) secondary germ cells. C and D) Germ cells (black asterisks) originating by asymmetric division from OSC (white asterisks) are accompanied by DR+ MDC (m), and also by CD8+ (F) and DR+ (G) T cells. E) Ig kappa light chain of immunoglobulins (Ig-kappa) is depleted in emerging germ cells. Adapted in part from [100] with permission, © Humana Press.

Figure 5

Model of OSC commitment for production of secondary germ cells. A) The uncommitted OSC (u-OSC) is present during sixth week of gestational age, prior to the arrival of primordial germ cells (pgc). B) Primordial germ cells invade OSC during seventh week and cause commitment of OSC (c-OSC) for production of secondary germ cells (sgc). C) The primordial germ cells degenerate and secondary germ cells are produced from OSC influenced by hormonal signaling and cellular signaling (MDC, Thy-1 pericytes, and T cells). The secondary germ cells enter ovarian cortex and differentiate into definitive oocytes (do). D) All OSC are influenced by systemic hormonal signals (HS), but only those influenced by CS undergo asymmetric division (ad) followed by symmetric division (sd) required for crossing over (co). Tadpole-like migrating secondary germ cells (m-sgc) leave OSC and enter the ovarian cortex. E) Origination of secondary germ cells from OSC by asymmetric division appears to require primitive (CD14+) MDC (P-MDC), activated pericytes (P) with a lack of suppressive neural signaling (NS-), activated (DR+) MDC (A-MDC) and activated (DR+) T cells (T). Adapted from [69] with permission, © Transworld Research Network.

Figure 6

Origin of new granulosa cells from OSC during the prime reproductive period in adult human ovaries. A) Panoramic view of ovarian surface and adjacent cortex. Dashed line indicates interface between TA and stroma of the ovarian cortex. osc and black arrow - ovarian stem cells; taf and black arrowhead - TA flap; white arrowhead - a lack of OSC above the TA; white arrow -bilaminar epithelial cord. B) Detail from (A) shows association of CK+ (brown color) fibroblasts (+fb,) with the TA flap surface (arrowhead), transition from mesenchymal to epithelial morphology (fb/se), and ovarian stem cells (osc, arched arrow). C) A parallel section to (B) showing numerous DR+ MDC (asterisks) in the TA flap. Note DR expression also in early OSC (arrow). D) Detail from (A) shows CK+ epithelial cord consisting of two layers of epithelial cells and lying between the ovarian cortex (ovc) and TA (ta). Note diminution of CK immunoexpression in TA fibroblasts (+/-fb). E) Epithelial cords (black arrows) fragmenting into granulosa cell nests (arrowheads in the upper ovarian cortex (uc). White arrow CK+ OSC associated with the TA with flap. F) Lower ovarian cortex (lc) with primordial follicles. Arrow indicates distance from the ovarian surface, dashed box indicates follicle shown in the inset. F29 indicates female age in years. Bar in (D), for (B-D). Panels A, B, D-F adapted from [11], © Antonin Bukovsky; panel C from [134], © Wiley-Liss, Inc. with permission.

Figure 7

Follicular renewal in adult human ovaries. A) Cytokeratin (CK) positive (brown color) cells of fibroblast type (fb) in tunica albuginea (ta) exhibit mesenchymal-epithelial transition into OSC (osc). Inset shows a transitory stage (fb/e). B) The CK+ epithelial nest (n) inside of the venule (v) in deep ovarian cortex, which extends an arm (a) to catch the oocyte (o, dashed line) from the blood circulation. e = endothelial cell. C) The nest body (n) and closing "gate". A portion of the oocyte (dashed line) still lies outside of the complex, and is expected to move inside (arched arrow). The oocyte contains intraooplasmic CK+ extensions from the nest wall (arrowheads), which contribute to the formation of CK+ paranuclear (Balbiani) body (asterisk). The oocyte nucleus is indicated by a dotted line. D) The occupied "bird's" nest type indicates a half way oocyte-nest assembly. CK indicates cytokeratin staining of primitive granulosa cells and ZP indicates zona pellucida expression in the assembling oocyte. E) Segments of OSC show cytoplasmic PS1 (meiotically expressed carbohydrate) expression. Asymmetric division of OSC gives rise to cells exhibiting nuclear PS1 (+ nuclei vs. - cell daughters) and descending from the OSC into tunica albuginea (ta). F) In tunica albuginea, the putative germ cells increase in size, show a symmetric division (black arrow) and exhibit development of cytoplasmic PS1 immunoexpression when entering (white arrow) the upper ovarian cortex (uc). G) Association of primary follicles (arrowhead) with the cortical epithelial crypt (ec). Dashed boxes indicate unassembled epithelial nests. Inset shows origination of germ-like cells among CK+ cells (CK) in epithelial crypt. Note ZP+ segment (white arrowhead) associated with unstained round cell nucleus (asterisk). H) Migrating germ cells with tadpole shape (dashed line), unstained nucleus (dotted line) and ZP+ staining of the intermediate segment (arrowhead). I) Some medullary vessels (v) show accumulation of ZP+ (dark color) degenerating oocytes with unstained nuclei (arrowheads). Arrow indicates ZP release. Adapted from [11], © Antonin Bukovsky.

Figure 8

Immune type cells influence commitment of OSC. Staining of the adult human OSC (osc), tunica albuginea (ta), and an adjacent cortex (ct) for CD14 of primitive MDC and HLA-DR of activated MDC, CD8 of cytotoxic/suppressor T cells, MHC class I heavy chain, and Thy-1 glycoprotein of pericytes, as indicated in panels. Large asterisks and dashed lines indicate putative germ cells. A) Primitive MDC associate with OSC (arrows) and accompany (arrowheads) origination of germ cells by asymmetric division of OSC (asterisks). B) Asymmetric division is also accompanied by extensions from T cell (arrowheads) into a putative germ cell daughter. C) Primitive MDC accompany (white arrowheads) symmetric division (s-s') of germ cells in tunica albuginea and their migration into the adjacent cortex (ct). D) Migrating tadpole-like germ cells are accompanied by activated MDC (open arrow), and HLA-DR material is apparent in the cytoplasm (solid arrow) and in the nuclear envelope (arrowhead). E) The germ cells associate with cortical vasculature (cv) strongly expressing MHC-I (arrows vs. arrowhead), enter and are transported by the bloodstream (F). Adapted from [87], with permission, © Blackwell Publishing, Oxford, UK.

Figure 9

Survey of follicular renewal in adult human ovaries. Follicular renewal in adult human ovaries is a two-step process based on mesenchymal-epithelial transition of tunica albuginea (ta) bipotent progenitor cells into OSC. A) Epithelial nests: Segments of the OSC directly associated with the upper ovarian cortex (uc) are overgrown with tunica albuginea, which forms a solid epithelial cord that fragments into small epithelial nests (en) descending into the lower ovarian cortex (lc) and associating with the blood vasculature. Initiation of this process may require cellular and other local signaling (CS & LS), possibly neural [87]. B) Germ cells: Under the influence of cellular signaling (CS) of ovary-committed MDC & T cells (OC-BMC) and hormonal signaling (HS), some OSC covering the tunica albuginea undergo asymmetric division and give rise to new germ cells (gc). The germ cells subsequently divide symmetrically and enter adjacent cortical blood vessels. During vascular transport, they are picked up by epithelial nests associated with vessels. D) The ovary-committed bone marrow cells originate from bone marrow (MDC) and from lymphoid tissues (T cells) carrying "ovarian" memory (om), which diminishes with utilization; when spent, the follicular renewal ceases, in spite of persisting hormonal signaling (Table  1).

Figure 10

Evolution of ovaries during developmental immune adaptation and their behavior during immune competence. A) Primordial germ cells imprint the OSC for production of secondary germ cells (see Figure  5B vs. 5A). B) Development of rete ovarii and lymphoid tissue (LT). Uncommitted MDC and T cells (UMT) saturate rete ovarii to be converted into ovary-committed MDC and T cells (OCMT). C) Secondary germ cells originate by asymmetric division of OSC under the influence of rete-derived OCMT and hormonal signaling. The ovary commitment is also transferred into draining lymphoid tissues (arched arrows). During the perinatal period immune competence (ic) is initiated, the ovarian memory (om) is built and the rete ovarii regresses. During childhood the OCMT is available but hormonal signaling is absent until menarche. E) During the prime reproductive period (from menarche to 38+/-2 years of age) OCMT and cyclic hormonal signaling cause cyclic formation of germ cell and renewal of primordial follicles. F) After the prime reproductive period the hormonal signalling persists but follicular renewal ceases due to the lack of OCMT. Reprinted from [132], with permission, © Bentham Science Publishers, Ltd.

Figure 11

Follicular selection. Selection of secondary (A-D) and preovulatory (dominant) follicles (E-G), and large antral follicle atresia (H-J) in the adult human ovary. Staining for Thy-1, HLA-DR (DR), MHC class I light chain (beta 2m) and CD68 of mature MDC, as indicated in panels. Dashed line in A indicates an "ovary-in-ovary" area exhibiting diminution of Thy-1 expression by stromal cells. B) detail from A. C and D are semi-parallel sections to B. Dashed line in E-J, follicular basement membrane. rf, resting follicles; gf, growing follicle; p, pericytes; e, endothelial cells; v, microvasculature in theca interna (t); vl, vascular layer adjacent to the follicular basement membrane; g, granulosa layer. Reprinted from [134] with permission, © Wiley-Liss.

Figure 12

Oocyte and parthenote development in vitro. A) The oocyte (o) development in OSC culture is accompanied by satellite (black arrow) and neuronal (white arrow) cells. Black arrowheads indicate organelles mowing from the satelite cell into the oocyte and white arrowhead indicates neuronal extension. Note ZP staining of fibro-epithelial cells (f) but no expression of ZP proteins at the oocyte surface (red arrowhead). Sperm associate (arrowheads) with fibroepithelial cells (B) but not with the oocytes (C). D and E) The parthenote shows a trophectoderm (te), blastocoele (bc), and inner cell mass (icm). (A) shows staining for ZP proteins, (B, C and D) are live cultures in phase contrast (PhC), (E) is a DAPI staining of the fixed culture. Adapted from [179], © Cambridge University Press, and unpublished observations.

Figure 13

In vitro developing oocytes supplied with organelles from fibroblasts resulting in fibro-oocyte hybrids, or by satellite cells produced by the oocytes and exploited for the progression of the oocyte growth. Images from time lapse photography (time in hours:minutes). A) In vitro developing oocytes (o) deficient in organelles (white arrow) can be joined (arrowhead) by a fibroblast (fb and white arrowhead). B) The optically dense organelles are supplied to the oocyte. C) Alternatively, the oocyte is supplied by adjacent satellite cell (s) with an extended tube (black arrowhead; see detail in inset). D) When completed, the tube disappears (inset) and the satellite is regressing (rs). E) In contrast, the fibroblast moves above the oocyte and releases organelles out (white arrowhead) of the regressing oocyte (ro). F) Subsequently, a fibro-oocyte (fbo) hybrid is formed exhibiting oocyte remnants (ro). Bar in A for A-F. Panels A-D adapted with permission from [9], © Wiley-Liss, Inc, and complemented with E and F.

Figure 14

Oocyte satellites in vitro. A) Early stage after oocyte (o) division producing the satellite (s) cell. Asterisks indicate additional small satellites. B) Oocyte growth and regressing satellite (rs) with vacuolization in the perinuclear space (arrowhead). C) Progressive oocyte growth and its separation (arrowhead) from the satellite remnants (sr). D) Large isolated oocyte exhibits germinal vesicle (gv) and a thick zona pellucida (zp) cytoplasmic membrane. Panels A-D adapted with permission from [9], © Wiley-Liss, Inc.

Figure 15

Differentiation of early oocytes and behavior of oocytes in secondary OSC cultures. A-C) Time lapse cinematography of oocyte development in secondary OSC culture. A) Early developing cell with a cytoplasmic tail (arrowhead). B) Multiple cytoplasmic eruptions (arrowheads). C) Development of the 50 micrometers oocyte-like cell (yellow arrowheads indicate cell surface, red arrowhead a polar body). Time in minutes':seconds". Movie segments are available in the Additional file 1, supplemental video S1. D-F) Day 3 secondary OSC culture stained for ZP (d3sc ZP) - 55 year old postmenopausal women three days afer seeding. D) Oocyte (o) with a "chain" of satellite cell (s), regressing satellites (rs), and satelite remnants (sr) interconnected by intercellular bridges (open white arrowheads) and surface ZP expression (open black arrowheads). The "leading" oocyte shows formation of the cell surface in the bridge (solid yellow arrowhead). Asterisk indicates cell nucleus and red arrowhead developing polar body. E) Two "leading" oocyte type cells with surface ZP expression, each of which shows expulsion of two polar bodies (red arrowheads) and retention of two pronuclei (asterisks). Yellow arrowheads indicate a line of separation from remnants of the "satellite" cells lacking surface ZP. F) Large oocyte (140 micrometers) with surface ZP expression (arrowhead), nucleus (asterisk), and germinal vesicle (gv).

Figure 16

Parthenotes in OSC cultures. A-C) Four cell embryo. D and E) morula. F and G) Blastocyst consisting of blastocoele (bc), trophectoderm (te) and inner cell mass (icm) releasing (arched arrow) DAZL+ embryonic stem cells (esc). Scale in C for A-C. A, C and F - Dazl staining, B, E and G - DAPI. Adapted with permission from [179], © Cambridge University Press.

Figure 17

Influence of the presence vs. absence of OSC in the ovary on ovarian cultures. Occurrence of OSC in pre- and postmenopausal ovaries (A-C); note a lack of adult primordial follicles in all samples. A) Staining for CK with hematoxylin counterstain shows CK+ OSC (arrowhead) in the ovary of 36 years old woman (F36). B) Ovary of 50 years old women without OSC. C) Activity of the TA (white arrowhead) with OSC in 55 year old ovary. The OSC is also present in the cortical crypt (yellow arrowhead) in the deeper cortex. (D-G) Phase contrast from live day 6 primary ovarian cultures (d6pc). D) Ovarian culture of a 50 year old female shows only narrow fibroblasts (note lack of OSC in panel B). E) Cluster of epithelial type cells in culture from a 55 year old female (note OSC in panel C). F) Detail from another cluster of epithelial cells shows small (30 micrometers) round oocyte type cell (arrowhead) with a prominent nucleus. G) Large (120 micrometers) round oocyte type cell with germinal vesicle (gv) and thickened plasma membrane (arrowhead).

Figure 18

Intravascular degenerating human oocytes. Degenerating oocytes in venules of ovarian medulla (A D and F) and uterine endocervical stroma (E) expressing zona pellucida proteins identified by MAb clones against ZP3, ZP4 and ZP 2 [198] as indicated in panels. MAb to ZP1 was kindly provided by Dr. Satish K Gupta. Arrowheads indicate unstained oocyte nuclei, arrow in F shows an arteriole, arrow in G shows ZP expression at the oocyte surface in a normal secondary follicle. F28-F38 indicate patients’ ages. Panels A and C adapted from [139] with permission, © Elsevier.

Figure 19

Snapshots from the collection of the OSC and ovarian biopsy from POF ovary by a laparoscopy. OSC are collected by scratching the ovarian surface with scissors (arrows in A) and with a brush (arrow in B). Ovarian biopsies (C and D) are collected from each ovary. For a complete video see Additional file 3, Video S2.