G C A T T A C G G C A T genes

Review The Crazy

Philippe Monget 1,*, Ken McNatty 2 and Danielle Monniaux 1

1 UMR INRAE-CNRS-IFCE-Université de Tours, 37380 Nouzilly, France; [email protected] 2 School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand; [email protected] * Correspondence: [email protected]

Abstract: From fetal life until senescence, the ovary is an extremely active tissue undergoing con- tinuous structural and functional changes. These ever-changing events are best summarized by a quotation attributed to Plato when describing motion in space and time—‘nothing ever is but is always becoming ... ’. With respect to the ovary, these changes include, at the beginning, the processes of follicular formation and thereafter those of follicular growth and atresia, steroidogenesis, maturation, and decisions relating to the number of mature that are ovulated for fertilization and the role of the . The aims of this review are to offer some examples of these complex and hitherto unknown processes. The ones herein have been elucidated from studies undertaken in vitro or from normal in vivo events, natural genetic mutations or after experimental inactivation of gene function. Specifically, this review offers insights concerning the initiation of follicular growth, pathologies relating to poly-ovular follicles, the consequences of premature loss of germ cells or oocytes loss, the roles of AMH (anti-Müllerian hormone) and BMP (bone morphogenetic protein) genes in regulating follicular growth and rate together with species differences in maintaining luteal function during . Collectively, the evidence suggests that the oocyte  is a key organizer of normal ovarian function. It has been shown to influence the phenotype of the  adjacent somatic cells, the growth and maturation of the follicle, and to determine the ovulation rate.

Citation: Monget, P.; McNatty, K.; When germ cells or oocytes are lost prematurely, the ovary becomes disorganized and a wide range Monniaux, D. The Crazy Ovary. of pathologies may arise. Genes 2021, 12, 928. https://doi.org/ 10.3390/genes12060928 Keywords: ovary; ; reactivation; primordial follicles; ovulation rate; unexpected; crazy; steroidogenesis Academic Editors: Ahmed Balboula and Annie Robic

Received: 18 May 2021 1. Introduction Accepted: 15 June 2021 The mammalian ovary has been, and continues to be, an extensively studied organ. Published: 18 June 2021 With respect to human health and well-being, significant challenges remain, including the restoration of fertility in women with polycystic syndrome; assisting those experiencing Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in an early depletion of their oocyte reserve and; for both human and animal conservation, published maps and institutional affil- new technologies to promote ovarian follicular growth in vitro in order to obtain mature iations. oocyte for fertilization and a successful pregnancy. Many reviews have already been written on all these aspects. Of interest is the fact that mammalian sometimes exhibit quite remarkable functional quirks, whether these are part of normal physiology or observed in nature by mutations or after experiments inducing functional modifications by inactivating or enhancing gene function. The purpose of this review is to offer a Copyright: © 2021 by the authors. few examples of the above to show that the ovary is a complex tissue to study with Licensee MDPI, Basel, Switzerland. sometimes unpredictable (“crazy?”) results. The presented examples address: the initiation This article is an open access article distributed under the terms and of follicular growth; the consequences of oocyte loss for somatic ovarian cells; pathologies conditions of the Creative Commons relating to poly-ovular follicles; the roles of AMH (anti-Müllerian hormone) and BMP (bone Attribution (CC BY) license (https:// morphogenetic protein) genes in regulating follicular growth and ovulation rate, and; some creativecommons.org/licenses/by/ species differences in maintaining luteal function during pregnancy. The intention of this 4.0/). review is not to be exhaustive, but to go beyond the scope of classic reviews.

Genes 2021, 12, 928. https://doi.org/10.3390/genes12060928 https://www.mdpi.com/journal/genes Genes 2021, 12, 928 2 of 13

1.1. The Initiation of Follicular Growth: The Hippo Signaling System Ovarian follicular formation takes place entirely within the ovigerous cords [1]. Pri- mordial follicles are formed sequentially from the base of the cords as each oocyte and single layer of epithelial cells separate by isolating themselves by a basement membrane. Thereafter, the first follicles that form are the first to grow and this occurs even before the full complement of primordial follicles have assembled [1]. In a classic series of studies, Peters et al. showed that follicles grow sequentially and continue to grow until they die or ovulate [2]. Moreover, this process is not interrupted from the time follicles are formed and continues throughout life, irrespective of reproductive status until death or until the primordial pool is depleted. It is not known whether the order at which follicular growth is initiated is determined by the order in which the follicles are formed. However, even if there is an ordered process, it can be disrupted. In 2013, Aaron Hsueh and colleagues published an important paper showing that when segments of ovaries (i.e., ovarian frag- mentation) are removed into culture, the initiation of primordial follicular growth can occur through the polymerization of actin and disruption of the Hippo signaling pathway in the [3]. This disruption causes the nuclear translocation of Yes-associated protein (YAP), which interacts with transcriptional-enhanced associated domain (TEAD) transcriptional factors. In turn, this leads to the expression of CCN (cystein-rich 61, con- nective tissue growth factor and nephroblastoma overexpressed) growth factors as well as BIRC (baculoviral inhibitors of apoptosis repeat containing) apoptosis inhibitors. This growth activation can be enhanced further by utilizing PI3K/AKT activators that act synergistically with the inhibition of Hippo pathway. This In Vitro Activation (IVA) approach has led to the delivery of a live offspring. Thus, it offers a practical solution for some patients who suffer from a premature ovarian insufficiency (POI). This IVA technology has also been used successfully in both Japan and China to treat POI. However, IVA is not a practical solution for more than 50% of POI patients who have few, if any, residual follicles in their ovaries (Figure1).

1.2. The Deletion of Omgc1 in Oocytes and Cyclic Ovarian Steroidogenesis in the Interstitium? The ovum mutant candidate gene 1 (Omcg1) encodes a nuclear zinc finger protein involved in pre-mRNA processing including splicing and transcription-coupled repair. Oocyte-specific inactivation of Omcg1 in early growing oocytes leads to reduced tran- scription, accumulation of DNA double-strand breaks as well as p63-dependent oocyte death. Mutant adult female mice are sterile with follicular growth arrested at the primary stage [4]. Remarkably, these mutant females, although sterile, exhibit vaginal cyclicity, with a normal onset of and even sexual receptivity, suggesting that there is a cyclical production of oestradiol. The evidence indicates that the Omcg1ocKO ovaries are dramat- ically remodeled with oocyte-depleted follicles able to express the main steroidogenic enzymes through an apparent cooperation between interstitial cells and granulosa cells (Figure2). These results are the first showing the possibility of an uncoupling between the cyclicity of sexual hormone secretion, and the follicular growth from small to preovulatory follicles. They also raise the possibility of internal clock genes within the somatic cells of the ovary. However, an alternative explanation might be a ‘cyclic-like’ positive-negative ovarian-pituitary feedback loop, but this requires further investigation.

1.3. Depletion of Oocytes before Puberty and the Fate of Granulosa Cells During the assembly of primordial follicles, significant numbers of the granulosa cells are recruited from the ovarian surface epithelium [1,5]. After the follicles have assembled, no new follicles form during the prepubertal or adult life. Therefore, the long-term via- bility of oocytes within primordial follicles is critical for reproductive success in later life. Although oocytes are equipped with an impressive array of repair mechanisms, their via- bility can be threatened by genetic mutations, X-irradiation, and chemo-toxic drugs [6–8]. In homozygous genetically-mutant mammals (e.g., Dazl; Fancd2; growth differentiation factor 9 (GDF9); bone morphogenetic protein 15 (BMP15)) where germ cells or oocytes die Genes 2021, 12, 928 3 of 13

without significant loss of the population [9], ovaries undergo significant, seemingly disorganized, restructuring with a diverse array of abnormal structures: these Genes 2021, 12, x FOR PEER REVIEW 3 of 14 can include the formation of intraovarian cords, ducts, clusters of endocrinologically-active cells and/or variable-sized internal or surface-visible solid tissues.

Figure 1.Figure Ovarian 1. Ovarian fragmentation, fragmentation, in vitroin vitro AktAkt stimulation stimulation and and auto-graftingauto-grafting promoted promoted follicle follicle growth growth in POI in (premature POI (premature ovarian ovarianinsufficiency) insufficiency) patients patients and andgenerate generatedd mature mature oocytes oocytes are ableable to to be be fertilized fertilizedin vitro in vitroleading leading to a pregnancy to a pregnancy and and a baby aftera baby after embryo transfer. transfer. Ovaries Ovaries removed removed under under laparosco laparoscopicpic were were cut cut into into strips, strips, the latterthe latter being being vitrified. vitrified. After After 3 thawing,thawing, strips were strips fragmented were fragmented into into small small cubes cubes (1–2 (1–2 mm mm3)) that disrupteddisrupted the the Hippo Hippo signaling signaling pathway, pathway, and treated and treated with Aktwith stimulators Akt stimulators (bpV (bpV(hopic) (hopic) and and740YP). 740YP). Forty-eight Forty-eight hour hourss later, cubes cubes were were autografted autograf underted under laparoscopic laparoscopic surgery surgery beneath beneathserosa of serosa Fallopian of Fallopian tubes. tubes. After After detection detection of of antral antral fo folliclesllicles viavia transvaginal transvaginal ultrasound, ultrasound, patients patients were treated were treated with with gonadotrophins,gonadotrophins, and mature and mature oocytes oocytes were were subjected subjected to to IVF IVF (in (in vi vitrotrofertilization) fertilization) before before cryopreservation of four-cell of four-cell stage stage .embryos. Patients Patients then received then received hormon hormonalal treatments treatments toto prepareprepare the the endometrium for implantation for implantation followed followed by transferring by transfer- ring of thawedof thawed embryos. embryos. Several Several babiesbabies have have been been born born using us thising techniquethis technique around around the world the ([3 world], with permission).([3], with permission).

− − 1.2. The DeletionIn Dazl ( of/ Omgc1) mice, in devoid Oocytes of theand RNA-bindingCyclic Ovarian protein, Steroidogenesis in and the follicular Interstitium? assembly is compromised. Consequently, follicles do not form, and the resulting somatic cellThe abnormalities ovum mutant may candidate display variable gene levels 1 (Omcg1 of endocrine) encodes activity a nuclear reminiscent zinc of finger that for protein involvedthe aforementioned in pre-mRNAOmgc1 processing (−/−) mice including but without splicing evidence and of pubertytranscription-coupled onset. repair. Oocyte-specificIn the GDF9 inactivationand BMP15 of mutantOmcg1 mice in early and sheep,growing normal oocytes populations leads to of reduced primordial transcrip- tion,follicles accumulation are assembled. of DNA However, double-strand after follicular breaks growth as well is as initiated p63-dependent and the epithelial- oocyte death. like granulosa cells become cuboidal, the absence of the oocyte-derived growth factors, MutantGDF9 adult and/or female BMP15, mice leads are to sterile the oocyte with continuing follicular to enlargegrowth but arrested without at a concomitantthe primary stage [4]. Remarkably,proliferation in these the numbers mutant offemales, granulosa although cells [10 ,sterile,11]. Subsequently, exhibit vaginal the oocytes cyclicity, die, with a normalleaving onset nodules of puberty of partially and differentiatedeven sexual granulosareceptivity, cells. suggesting The nodules that are there sometimes is a cyclical production of oestradiol. The evidence indicates that the Omcg1ocKO ovaries are dramati- cally remodeled with oocyte-depleted follicles able to express the main steroidogenic en- zymes through an apparent cooperation between interstitial cells and granulosa cells (Fig- ure 2). These results are the first showing the possibility of an uncoupling between the cyclicity of sexual hormone secretion, and the follicular growth from small to preovula- tory follicles. They also raise the possibility of internal clock genes within the somatic cells of the ovary. However, an alternative explanation might be a ‘cyclic-like’ positive-nega- tive ovarian-pituitary feedback loop, but this requires further investigation.

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observed in clusters together with solid luteal-like bodies and large cysts often with endocrineobserved activities in clusters (e.g., together oestradiol, with inhibin, solid luteal-like progesterone). bodies and large cysts often with endocrine activities (e.g., oestradiol, inhibin, progesterone).

Figure 2. Summary of the ovarian and reproductive features of Omcg1ocKo females. Oocyte death and arrest of folliculogene- Figuresis at the 2. Summary primary follicle of the ovarian stage, leading and reproductive to a kind of features premature of Omcg1 ovarianocKo females. insufficiency Oocyte (POI), death were and observed arrest of folliculogene- in Omcg1ocKo ocKo sisfemales.Figure at the 2. primaryThe Summary of somatic the stage, ovarian compartment leading and reproductive to a was kind remodeled, of prematurefeatures allowingof Omcg1 ovarian theocKo insufficiency females. production Oocyte of (POI), oestradiol death were and (E2),observed arrest which of infolliculogen- mightOmcg1 occur females.alongesis at a the two-cellThe primary ovarian compartment follicle somatic stage, compartment scheme leading similar to was a kindto remodeled, what of premature is found allowing betweenovarian the production insufficiency theca cells of and oestradiol(POI), granulosa were (E2), observed cells which in preovulatoryin might Omcg1 occurocKo alongfollicles.females. a two-cell Surprisingly, The ovarian compartment somatic despite compartment the scheme absence similar of was cyclic to remodeled, what follicular is found allowing growth between and the production then theca of cells preovulatory of and oestradiol granulosa follicles, (E2), cells whichOmcg1 in preovulatory mightocKo females occur follicles.displayedalong a Surprisingly, two-cell features compartment of sexualdespite cyclicity the scheme absence as similar wild-type of cyclic to what follicularmice (adaptedis foun growthd between from and [4 then]). theca of cells preovulatory and gran follicles,ulosa cellsOmcg1 in preovulatoryocKo females ocKo displayedfollicles. featuresSurprisingly, of sexual despite cyclicity the absence as wild-type of cyclic mice follicular (adapted growth from and [4]). then of preovulatory follicles, Omcg1 females displayed features of sexual cyclicityThe as FANCD2 wild-type protein mice (adapted is normally from [4]). expressed in both oocytes and granulosa cells andThe plays FANCD2 an important protein role is innormally DNA repair expressed and meiotic in both maturation. oocytes and However, granulosa in cells the andFancd21.3. playsDepletion-KO anmice, importantof Oocytes some before primordial role inPuberty DNA follicles and repair the assembleFate and of meiotic Granulosa around maturation. Cells birth. All However, oocytes, but in the not Fancd2the granulosaDuring-KO mice, the cells, someassembly die primordial within of primordial the follicles first follicles, three assemble months significant around of life. birth.numbers Subsequently, All of oocytes, the granulosa extensive but not theintraovariancells granulosa are recruited remodeling cells, from die occurswithinthe ovarian with the firstsexsurface cords three epithelium and months epithelial of[1,5]. life. tubules After Subsequently, becomingthe follicles increasingly extensivehave as- intraovarianprominentsembled, no with remodeling new age. follicles Thereafter, occurs form with during tumors sex cordsthe with prepubertal and multiple epithelial or phenotypes adult tubules life. becoming Therefore, develop, increasingly withthe long- some prominentfulfillingterm viability the with criteria of age. oocytesThereafter, of malignant within primordial tumors carcinomas with follicles multiple (Figure is critical3 phenotypes)[ 9]. for One reproductive interpretation develop, withsuccess of some thisin fulfillingphenomenonlater life. theAlthough criteria is that oocytes the ofepithelial-like malignant are equipped carcinomas granulosa with an (Figure cells impressive have3)[ reverted9]. array One of to interpretation repair their fetal mechanisms, phenotype of this phenomenonandtheir form viability cords is thatcan in an thebe attempt threatened epithelial-like to locate by granulosagenetic germ cells.mutations, cells have X-irradiation, reverted to their and fetal chemo-toxic phenotype anddrugs formCollectively, [6–8]. cords In inhomozygous thesean attempt data suggestgene to locatetically-mutant that germ the cells. epithelial-derived mammals (e.g., granulosaDazl; Fancd2 cells; growth associated dif- withferentiationCollectively, oogonia factor or withthese 9 (GDF9) oocytes data suggest; bone in primordial morphogenetic that the epithelial-derived follicles protein retain 15 a (BMP15) high granulosa level) where of cells plasticity germ associated cells and withhaveor oocytes oogonia the potential die or without with to form oocytes significant ovarian in primordial cancersloss of ifth the folliclese granulosa viability retain ofcell the a population high oocytes level is of at[9], plasticity risk. ovaries However, un- and haveoncedergo the primordial significant, potential follicles toseemingly form startovarian di tosorganized, grow cancers and if restructuring the viability epithelial-derived ofwith the a oocytes diverse follicular isarray at risk. cellsof abnormal However, develop onceastructures: granulosa primordial these cell folliclesphenotype,can include start extensivethe to growformation andintraovarian theof in epithelial-derivedtraovarian modeling cords, still ducts, follicular occurs clusters but cells without of develop endo- the aappearancecrinologically-active granulosa cell of intraovarian phenotype, cells and/or sex extensive cords variable-sized and/or intraovarian epithelial-tubules. internal modeling or surface-visible still occurs butsolid without tissues. the appearanceIn Dazl of (− intraovarian/−) mice, devoid sex cordsof the and/or RNA-binding epithelial-tubules. protein, oogenesis and follicular as- 1.4.sembly Poly-Ovular is compromised. Follicles: FromConsequently, Physiology follic to Environmentallyles do not form, Induced and the Pathology resulting somatic 1.4.cell Poly-Ovular abnormalitiesAlthough aFollicles: single may display oocyte From Physiology variable per follicle levels to Environmentally is of generally endocrine the activity Induced rule in reminiscent Pathology mammals asof that in other for vertebrates,the aforementionedAlthough poly-ovular a single Omgc1 oocyte follicles (−/− per) mice are follicle but not without uncommonis generally evidence inthe the of rule puberty ovaries in mammals onset. of marsupials as in other and vertebrates,variousIn the eutherian GDF9 poly-ovular and mammals BMP15 follicles includingmutant are notmice humans. uncommon and sheep, In thenormal in the opossumovaries populations ovary, of marsupials of some primordial follicles and variouscanfollicles contain eutherianare assembled. up to a mammals 100 However, oocytes including per after follicle, follicul humans. mostar growth of In them the is opossuminitiated degenerating and ovary, the by epithelial-like some atresia follicles during canfolliculargranulosa contain growth cells up tobecome a [12 100].oocytes Thecuboidal, percentage per the follicle, absence of poly-ovular most of the of oocyte-derived them follicles degenerating is quite growth by variable atresiafactors, between duringGDF9 follicularspeciesand/or andBMP15, growth between leads [12]. individuals to The the percentageoocyte within continuing of species. poly-ovular to It enlarge is particularly follicles but without is high quite ina concomitant variable the ovaries between of pro- cats species(upliferation to 4% and in of between the follicles) numbers individuals and of dogs granulosa (up within to 14%cells species. of [10,11]. follicles), It is Subsequently, particularly each poly-ovular high the inoocytes the follicle ovaries die, containing leaving of cats (upbetweennodules to 4% 2of of and partially follicles) 17 oocytes differentiated and dogs [13,14 (up]. In togranulos humans, 14% ofa follicles), poly-ovularcells. The each nodules follicles poly-ovular are and sometimes polynuclear follicle containingobserved oocytes betweenarein clusters found 2 and in together 98% 17 oocytes of with 18- to [solid13 52-year-old,14 luteal-like]. In humans, women bodies poly-ovular and and represent large follicles cysts between often and polynuclearwith 0.06% endocrine and 2.44% oocytes ac- of arethetivities found total (e.g., follicular in 98%oestradiol, of population 18- toinhibin, 52-year-old of progesterone). the ovary women [15 and]. In represent poly-ovular between follicles, 0.06% the andquality 2.44% of the of theoocytes totalThe is follicular FANCD2 often heterogeneous. population protein is normally of Some the ovary poly-ovular expressed [15]. In in follicles poly-ovular both oocytes are able follicles, and toovulate granulosa the quality but cells generally, of and the oocytesatplays most an is a important oftensingle heterogeneous. oocyte role is in fertilized DNA Some repair [14 poly-ovular]. and meiotic follicles maturation. are able However, to ovulate in but the generally, Fancd2- atKO most mice,Three a single some possible oocyteprimordial modes is fertilized follicles of formation [assemble14]. of poly-ovular around birth. follicles All oocytes, have been but not suggested: the granu- the subsequentlosaThree cells, possibledie division within modes ofthe polynuclear first offormation three months oocytes, of poly-ovular of thelife. fusion Subsequently, follicles of previously have extensive been separate suggested: intraovarian follicles, the subsequentandremodeling the initial divisionoccurs failure with of of polynuclearsex separation cords and of oocytes, epithelial oocytes the by tubules follicularfusion becoming of cells previously in increasingly germ separate cell cysts prominent follicles, during andfollicularwith the age. initial formation.Thereafter, failure The oftumors separation first hypothesiswith multiple of oocytes is documented phenotypes by follicular indevelop, humans cells in with germ with some the cell observationfulfilling cysts during the of follicularoocytescriteria of containing formation. malignant twoThe carcinomas firstgerminal hypothesis (Figure vesicles is 3) documented (0.4%[9]. One of growinginterpretation in humans follicles, withof this from the phenomenon observation [15]) and with is of Genes 2021, 12, 928 5 of 13

the recovery and in vitro fertilization of bin-ovular complexes, in which the two oocytes are included in the same [16–18]. Concerning the second hypothesis, a few morphological studies in humans have suggested that bin-ovular follicles might be formed by the fusion of two nearby primordial or primary follicles [19,20]. Whatever the origin of Genes 2021, 12, x FOR PEER REVIEW 5 of 14 the bin-ovular complexes, the successful fertilization of both oocytes and the development of dizygotic twins was an exception [21].

FigureFigure 3. 3. RepresentativeRepresentative micrographs micrographs of of ovary ovary sections sections from fromFancd2-WT Fancd2-WT(A) ( andA) andFancd2-KO Fancd2-KO (B–J) (Bmice–J) mice at 2.5 at ( 2.5A–C (A),– 5–9C), 5–9(D– (ID) –andI) and 12 ( 12J) (monthsJ) months of ofage age after after birth. birth. Note Note the the appearance appearance of of primordial primordial(arrow) (B (arrow)), preantral (B), preantral (arrowhead) (arrowhead) (B) and (B antral) and antral(C) follicles (C) follicles in ovaries in ovaries of Fancd2-KO of Fancd2-KO mice similar miceto that similar in WT to that mice in WT(A).mice Common (A). Common abnormal abnormal ovarian ovarian phenotypes phenotypes in Fancd2-KO in Fancd2-KO mincemince include: the include:formation the formationof numerous of numerous sex (arrows) cords (arrows) (D); tubules (D); tubules (high (high magnification magnification inset, inset, arrow) arrow) (E); invag- (Eination); invagination of ovarian of ovarian surface surface epithelium epithelium (high (high magnification magnification inset, inset, arrow) arrow) (F); (F );cystic cystic papillary papil- hyper- laryplasia hyperplasia (high magnification (high magnification inset, ( inset,G); luteomas (G); luteomas containing containing large large areas areas of cells of cells of spongy of spongy appearance appearance(high magnification (high magnification inset, (H inset,); cystadenocarcinoma (H); cystadenocarcinoma whereby whereby mitotically-active mitotically-active cells cells are invading invadingextraovarian extraovarian fat cells fat High cells Highmagnification magnification inset, inset, (I) (and;I) and; large large areas areas containing containing numerous numerous tumour tumourphenotypes phenotypes of epithelial of epithelial origin, origin, including including adenocarcinomas adenocarcinomas (arrows) (arrows) ( J). ReproducedReproduced with with permis- permissionsion from fromUPV/EHU UPV/EHU Press Press from from [9]. [ 9].

AbnormalCollectively, germ-cell these cyst data breakdown suggest that and the assembly epithelial-derived of individual gran oocytesulosa with cells pre- associated granulosawith oogonia cells inor thewith ovigerous oocytes cordsin primordial of the fetal follicles or neonatal retain ovary a high is the level most of likelyplasticity and have the potential to form ovarian cancers if the viability of the oocytes is at risk. However, once primordial follicles start to grow and the epithelial-derived follicular cells develop a granulosa cell phenotype, extensive intraovarian modeling still occurs but without the appearance of intraovarian sex cords and/or epithelial-tubules.

1.4. Poly-Ovular Follicles: From Physiology to Environmentally Induced Pathology Although a single oocyte per follicle is generally the rule in mammals as in other vertebrates, poly-ovular follicles are not uncommon in the ovaries of marsupials and var- ious eutherian mammals including humans. In the opossum ovary, some follicles can con- tain up to a 100 oocytes per follicle, most of them degenerating by atresia during follicular growth [12]. The percentage of poly-ovular follicles is quite variable between species and

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hypothesis to explain the formation of most poly-ovular follicles. Intricate regulation of gene expression, including the oocyte-specific Figla [22], Nobox [23], and Taf4b [24] transcription factors, is critical for these processes in the mouse. The oocyte-specific secreted factors BMP15 and GDF9 might participate as well in controlling follicular formation, since the formation of poly-ovular follicles has been observed in Bmp15−/− Gdf9+/− mice [25]. In humans and sheep, BMP15 is not expressed until the follicle is formed and begins to grow, but it can be speculated that BMP15 secreted by the first growing follicles may activate BMPR1B (bone morphogenetic protein receptor type 1B) signaling in the ovigerous cords and influence the subsequent formation of follicles. In the mouse, somatic cell- derived growth factors such as activin/follistatin and neurotrophins also influence cyst breakdown [26–28], and the disintegrin Adam10 has been shown to govern the recruitment of the pre-granulosa cells in cysts [29]. The actions of these various factors converge to modulate the Jagged/Notch signaling pathway in germ cell cysts. Particularly, the Notch2 gene encoding a Notch receptor in pre-granulosa cells orchestrates cyst breakdown, postnatal apoptosis of oocytes and primordial follicle assembly [30] and the oocyte-specific secretory proteins Jagged1 (a Notch ligand), Gdf9 and Bmp15 activate the Notch signaling pathway in pre-granulosa cells [31]. All these observations highlight the importance of crosstalk between germ cells and pre-granulosa cells for the formation of primordial follicles [32] (Figure4). They suggest that the formation of poly-ovular follicles could result Genes 2021, 12, x FOR PEER REVIEW 7 of 14 from local disequilibrium of the concentration of factors involved in this crosstalk in the fetal or neonatal ovaries of mammals.

FigureFigure 4. 4. SchematicSchematic representation representation of of some some mechanisms mechanisms currently currently kn knownown to to regulate regulate germ germ cell cell cyst cyst breakdown breakdown and and follicle follicle assemblyassembly in in the the fetal fetal or or neonatal neonatal ovary. ovary. A A germ germ cell cell cyst cyst is is represented represented (delineated (delineated by by aa dotted dotted line), line), containing containing oocytes oocytes (blue round cells) and pre-granulosa cells (small pink cells). Some known interactions between germ and somatic cells (blue round cells) and pre-granulosa cells (small pink cells). Some known interactions between germ and somatic cells participating to these processes are zoomed in the right part of the figure (adapted from [32]). participating to these processes are zoomed in the right part of the figure (adapted from [32]).

TheThe fetal fetal gonad gonad synthesizes synthesizes steroids steroids and and intra-ovarian intra-ovarian oestradiol oestradiol likely likely participates participates inin the the physiological physiological mechanisms mechanisms regulating regulating follicle follicle assembly assembly [32]. [32]. Oestrogens Oestrogens can can control control follicularfollicular formation formation through through mechanisms mechanisms involving involving members members of of the the TGF- TGF-ββ familyfamily such such as as activinactivin (monkey: (monkey: [33]) [33]) and and BMP2 BMP2 (hamster: (hamster: [3 [344]).]). Whether Whether between-species between-species differences differences in in intra-ovarianintra-ovarian concentrations concentrations at at the the time time of of fo follicularllicular formation formation can can explain explain differences differences in in thethe percentages percentages of of poly-ovular poly-ovular follicles follicles observed observed between speciesspecies andand betweenbetween individualsindividu- alswithin within species species remains remains hypothetical, hypothetical, however. howeve Asr. As first first observed observed in Floridain Florida alligators alligators [35], [35],pharmaceutical pharmaceutical and environmentaland environmental oestrogens, oestrogens, such assuch phytoestrogens as phytoestrogens or pesticides or pesticides with es- withtrogenic estrogenic action, action, can induce can induce the formation the formation of poly-ovular of poly-ovular follicles infollicles the ovaries in the of ovaries mammals. of mammals. Particularly, early-life exposure to synthetic oestrogens (diethylstilbesterol) or environmental pollutants with oestrogen-like activity (genistein, zearalonone, diethylhex- ylphthalate) have been shown to impair the assembly of primordial follicles [36–39]. The presence of multiple poly-ovular follicles in mouse ovaries exposed to diethylhex- ylphthalate is associated with a decrease in the gene and protein expression of the oestra- diol receptor Esr2 and components of Notch signaling [39]. Thus, deregulation of the mechanisms of germ cell cyst breakdown and assembly of individual oocytes with pre- granulosa cells in the ovigerous cords by oestrogen compounds results in the establish- ment of a pathological ovarian reserve of poly-ovular primordial follicles.

1.5. Differences in Ovarian Follicular Characteristics between Species 1.5.1. From Teleost to Tetrapod The female reproductive system is different in several ways between teleost and tetrapod. In teleost (i.e., in most of them), fertilization is external, whereas in tetrapod it is internal with the exception of amphibians. This is most likely the reason why the genomes of tetrapods, and specifically mammals, have lost several genes encoding the proteins in- volved in sperm/egg interactions [40], including the ZP (zona pellucida) [41]. Indeed, these proteins constitute a species barrier important in water where gametes of several species are susceptible to being mixed, this barrier being less necessary when fertilization is internal. Secondly, most teleost ovulate tens or even hundreds of thousands of oocytes per ovulation. It is most likely for this evolutionary reason (a sort of evolutionary residue?) that mammalian ovaries still constitute a stock of several thousand oogonia during fetal

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Particularly, early-life exposure to synthetic oestrogens (diethylstilbesterol) or environmen- tal pollutants with oestrogen-like activity (genistein, zearalonone, diethylhexylphthalate) have been shown to impair the assembly of primordial follicles [36–39]. The presence of multiple poly-ovular follicles in mouse ovaries exposed to diethylhexylphthalate is associated with a decrease in the gene and protein expression of the oestradiol receptor Esr2 and components of Notch signaling [39]. Thus, deregulation of the mechanisms of germ cell cyst breakdown and assembly of individual oocytes with pre-granulosa cells in the ovigerous cords by oestrogen compounds results in the establishment of a pathological ovarian reserve of poly-ovular primordial follicles.

1.5. Differences in Ovarian Follicular Characteristics between Species 1.5.1. From Teleost to Tetrapod The female reproductive system is different in several ways between teleost fish and tetrapod. In teleost (i.e., in most of them), fertilization is external, whereas in tetrapod it is internal with the exception of amphibians. This is most likely the reason why the genomes of tetrapods, and specifically mammals, have lost several genes encoding the proteins involved in sperm/egg interactions [40], including the ZP (zona pellucida) [41]. Indeed, these proteins constitute a species barrier important in water where gametes of several species are susceptible to being mixed, this barrier being less necessary when fertilization is internal. Secondly, most teleost ovulate tens or even hundreds of thousands of oocytes per ovulation. It is most likely for this evolutionary reason (a sort of evolutionary residue?) that mammalian ovaries still constitute a stock of several thousand oogonia during fetal life, this stock disappearing largely before puberty. However, at the time of ovulation, mammalian ovaries ovulate significantly fewer oocytes than teleost, from 1 in women, cows and mares, between 2 and 5 to 6 in ewes (see below), and between 15 and 25 in sows.

1.5.2. Between Pig and Cattle Porcine ovaries contain a large number of growing antral follicles compared to those of cattle, sheep, women, and even dogs, cats, and rodents. The ovulation rate of sows can exceed 20, with some ovulating more than 50 follicles per cycle. Indeed, the surface of the sow’s ovary looks similar to a bunch of grapes. In part, this is due to the fact that the promoter of the porcine AMH gene is much less active than the bovine one, with the antral follicles of the pig secreting very low levels of the anti-Müllerian hormone (AMH) compared to those of the cow. Indeed, in mammalian females, AMH plays an inhibitory role in the recruitment of primordial ovarian follicles, the growth of small preantral follicles, and in modulating the sensitivity of antral follicles to FSH (follicle stimulating hormone), thereby restricting preovulatory follicular maturation [42–44]. In the pig, the low levels of AMH have very little effect in inhibiting the recruitment of primordial follicles, thereby leading to a larger number of growing follicles and a higher ovulation rate, compared to that in the cow (Figure5)[45].

1.5.3. Between Different Sheep Breeds: The Role of the BMP Family Influencing Ovulation Rate Mammals have evolved a number of strategies to limit the number of ovulated oocytes. Members of the BMP growth factor family have been identified as crucial factors in regulating ovulation rate in mammals [46] This role was revealed by the identification of several loss of function mutations in the oocyte-specific expression of GDF9 and especially BMP15 and the BMPR1B receptor [47,48]. Ewes carrying null-alleles of GDF9 or BMP15 in the heterozygous state have increased ovulation rates, whereas homozygous mutants are sterile. In contrast, both heterozygous and homozygous ewes, with a partial loss of function in the BMPR1B gene, hyper-ovulate (3 to 10 oocytes ovulated per cycle, compared to 1 or 2 in the wild-type). Another gene located on X chromosome (different from the BMP15 gene), yet to be identified, has also been shown to be in involved in an increase Genes 2021, 12, x FOR PEER REVIEW 8 of 14

life, this stock disappearing largely before puberty. However, at the time of ovulation, mammalian ovaries ovulate significantly fewer oocytes than teleost, from 1 in women, cows and mares, between 2 and 5 to 6 in ewes (see below), and between 15 and 25 in sows.

1.5.2. Between Pig and Cattle Porcine ovaries contain a large number of growing antral follicles compared to those of cattle, sheep, women, and even dogs, cats, and rodents. The ovulation rate of sows can exceed 20, with some ovulating more than 50 follicles per cycle. Indeed, the surface of the sow’s ovary looks similar to a bunch of grapes. In part, this is due to the fact that the promoter of the porcine AMH gene is much less active than the bovine one, with the antral follicles of the pig secreting very low levels of the anti-Müllerian hormone (AMH) com- Genes 2021, 12, 928 pared to those of the cow. Indeed, in mammalian females, AMH plays an inhibitory8 of 13 role in the recruitment of primordial ovarian follicles, the growth of small preantral follicles, and in modulating the sensitivity of antral follicles to FSH (follicle stimulating hormone), therebyin ovulation restricting rate when preovulatory it is mutated follicular at both maturation the homozygous [42–44]. and In heterozygous the pig, the statelow levels [49]. of AMHSurprisingly, have very mutations little effect in the in three inhibitingBMP15 the, BMPRIB recruitmentand the of unknownprimordial X-linked follicles, genes thereby leadingare additive, to a larger ewes carrying number the of threegrowing alleles follicles at the heterozygousand a higher stateovulation exhibiting rate, ovulationcompared to thatrates in >12 the [50 cow]. (Figure 5) [45].

FigureFigure 5. 5. DifferencesDifferences between between the the bovine bovine and and porcine porcine species species in in te termsrms of of anti-Müllerian anti-Müllerian horm hormoneone (AMH) (AMH) production production that couldthat couldexplain explain the regulation the regulation of ovulation of ovulation number. number. We propose We propose this this model model to explain to explain a potential a potential role role of ofAMH AMH in inthe the regu- lationregulation of the ofovulation the ovulation rate in rate porcine in porcine compared compared to bovine to bovine ovaries. ovaries. Porcine Porcine AMHAMH promoterpromoter and porcine and porcine granulosa granulosa cells are lesscells sensitive are less to sensitive bone morpho to bonegenetic morphogenetic protein (BMP) protein st (BMP)imulation, stimulation, leading leadingto a low to production a low production of AMH of by AMH porcine by porcine compared tocompared bovine growing to bovine antral growing follicles. antral As follicles. AMH Asis known AMH is to known be an to inhibitor be an inhibitor primordial primordial follicle follicle activation, activation, this low this lowlevel of AMH in the pig would lead to a huge number of growing follicles in the porcine compared to the bovine species. The level of AMH in the pig would lead to a huge number of growing follicles in the porcine compared to the bovine species. reduced BMP sensitivity of granulosa cells and the low intra-follicular AMH concentrations of antral follicles could con- The reduced BMP sensitivity of granulosa cells and the low intra-follicular AMH concentrations of antral follicles could tribute to sensitizing granulosa cells to FSH (follicle stimulating hormone), resulting in a high follicular survival rate in contribute to sensitizing granulosa cells to FSH (follicle stimulating hormone), resulting in a high follicular survival rate in the cohort of terminally developing follicles and a higher ovulation rate in the porcine, compared to the bovine species the cohort of terminally developing follicles and a higher ovulation rate in the porcine, compared to the bovine species (from [45], with permission). (from [45], with permission).

In most of these models, the preovulatory follicles are smaller in diameter in “mutants” compared with wild-type ewes. In most mammals, (e.g., human, sheep, pig, deer, rodents) both BMP15 and GDF9 are synthesized exclusively from oocytes. However, a notable exception is the pig, where GDF9 is also synthesized in granulosa cells [51]. Unexpectedly, it turns out that it is the oocytes and not the gonadotrophins that have a major influence on the species differences in the ovulation rate. In contrast to BMP15 and GDF9, the BMPR1B gene is expressed in both oocytes and the somatic cells within the ovary. It seems that the BMP system, and the relative levels of BMP15 and GDF9, determine the stage at which follicular maturation occurs. Moreover, as all the identified mutations are complete or have a partial loss of function, one can consider these mutations as having “brake-release” functions, thereby resulting in follicles ready to ovulate at smaller diameters. Despite these follicles ovulating at smaller diameters, it remains to be determined whether follicular Genes 2021, 12, 928 9 of 13

growth occurs at a different rate. Importantly, these mutations have no consequences on the capacity of oestradiol secretion by granulosa cells ([52] for Booroola mutants [53] and the heterozygous BMP15 mutant), we can hypothesize that the increase in the ovulation rate requires a sufficient number of granulosa cells to secrete enough oestradiol necessary for the induction of the GnRH surge [54] (Figure6). Moreover, after ovulation, the number of steroidogenic luteal cells was found to be similar between the Booroola mutants, which is consistent with the finding that there were no genotype differences in progesterone Genes 2021, 12, x FOR PEER REVIEW secretion [55]. It is worth noting that partial inhibition of BMP15 and/or GDF9 bioactivity10 of 14 in cattle [56] can also influence the ovulation rate and that GDF9 mutations in humans have been associated with dizygotic twinning [57].

Figure 6. Poly-ovulation in ewes carrying loss of function mutations in the ovarian BMP system. It seems that BMP15 Figureand GDF9 6. Poly-ovulation determine the in stageewes carrying at which loss follicular of function maturation mutations occurs. in the In ovarian the case BMP of loss system. of function, It seems onethat canBMP15 consider and GDF9these mutationsdetermine asthe having stage at “brake-release” which follicular functions, maturation thereby occurs resulting. In the incase follicles of loss ready of fu tonction, ovulate one at can smaller consider diameters. these mutationsAdditionally, as having as these “brake-release” mutations have functi no consequencesons, thereby resulting on the capacity in follicles of oestradiol ready to ovulate secretion at bysmaller granulosa diameters. cells ([ Addi-52] for tionally, as these mutations have no consequences on the capacity of oestradiol secretion by granulosa cells ([52] for Boo- Booroola mutants; [53] for heterozygous BMP15 mutant), we can hypothesize that the increase in the ovulation rate is due roola mutants; [53] for heterozygous BMP15 mutant), we can hypothesize that the increase in the ovulation rate is due to to the need to have a sufficient number of granulosa cells to secrete enough oestradiol necessary for the induction of the the need to have a sufficient number of granulosa cells to secrete enough oestradiol necessary for the induction of the GnRHGnRH surge surge [54]. [54]. 1.6. Why Are There So Many Luteotrophic Factors in Mammals? 1.6. Why Are There So Many Luteotrophic Factors in Mammals? Most of the elements involved in ovarian function in mammals have arisen in ver- tebrates,Most (e.g.,of the pituitary elements involved in ovarian and their function receptors), in mammals and the have follicular arisen in stages verte- of brates,growth (e.g., are morepituitary or less gonadotropins conserved in and mammals. their receptors), However, and a molecular the follicular mechanism stages of in- growthvolving are mammalian more or less ovaries conserved is radically in ma differentmmals. between However, species, a molecular namely mechanism the luteotrophic in- volvingfactors secretedmammalian by the ovaries young is embryo radically at thedifferent start ofbetween gestation. species, These namely factors arethe criticalluteotro- for phicpreventing factors thesecreted corpus by luteum the young from embryo disappearing at the bystart apoptosis, of gestation. which These would factors be detrimental are criti- calto thefor survivalpreventing of thethe embryo.corpus luteum These factors from disappearing are: by apoptosis, which would be detrimental to the survival of the embryo. These factors are: • the human chorionic gonadotrophin (hCG) in women, which binds to the LH receptor. • • thethe human trophoblastin chorionic (IFN gonadotrophinτ) in ruminants, (hCG) which in women, impairs which the secretionbinds to the of theLH PGF2recep-α tor. factor by the endometrium. •• thethe trophoblastin different forms (IFN of prolactinτ) in ruminants, in rodents, which which impairs inhibit the the secretion expression of the of PGF2 the proges-α lu- teolysisterone metabolizingfactor by the endometrium. 20-α-hydroxysteroid dehydrogenase (20αHSD) (Figure7,[58]). • the different forms of prolactin in rodents, which inhibit the expression of the pro- gesterone metabolizing 20-α-hydroxysteroid dehydrogenase (20αHSD) (Figure 7, [58]). This evolutionary incongruity is in reality most likely not due to the ovary itself, but to the placenta, which is an organ that has evolved rather rapidly between mammals. It is very probable that the ovarian corpus luteum, in order to ensure its maintenance two weeks after ovulation in large mammals, expresses the receptor adapted to the luteotro- phic factors expressed by the placenta of the species in question.

Genes 2021, 12, x FOR PEER REVIEW 11 of 14

Genes 2021, 12, 928 10 of 13 Genes 2021, 12, 928 10 of 13

Figure 7. Evolution and diversity of non-steroidal luteotrophic factors in eutherians. In primates, duplication of the (LH) gene generated chorionic gonadotropins (CG), which are expressed by trophoblast and impair corpus luteum lysis in the first months of pregnancy. In rodents, pituitary prolactin (PRL) released in response to coitus inhibits 20-hydroxysteroid dehydrogenase (20α-HSD), this function being subsequently assumed by placental lactogens (PL-1 and PL-2). In ruminants, duplication of the IFNW gene generated interferon-τ (IFNT) secreted by the blastocyst and acting on the endometrium to inhibit the prostaglandin F2α (PGF2α) luteolytic signal. In elephants, the PRL gene expressed in the placenta is responsible for pregnancy maintenance by accessory corpora lutea. In red, proteins born after gene duplication (adapted from [58]).

This evolutionary incongruity is in reality most likely not due to the ovary itself, but Figure 7. Evolution and diversity ofto the non-steroidal placenta, which luteotrophic is an organ that factors has evolved in eutherians. rather rapidly In primates, between mammals. duplication It of the luteinizing hormone (LH) gene generatedis very chorionicprobable that gonadotropins the ovarian corpus (CG), luteum, which in are order expressed to ensure its by maintenance trophoblast two and impair corpusFigure luteum 7. Evolution lysis in and the firstdiversity monthsweeks of non-steroidal of after pregnancy. ovulation luteotrophic In in large rodents, mammals, pituitary factors expresses in prolactin eutherians. the receptor (PRL) In adapted primat released toes, the induplication luteotrophic response toof coitusthe lute- factors expressed by the placenta of the species in question. inhibitsinizing 20-hydroxysteroid hormone (LH) gene dehydrogenase generated chorionic (20α-HSD), gonadotropins this function (CG), being wh subsequentlyich are expressed assumed by trophoblast by placental and lactogens impair cor- (PL-1pus and luteum PL-2). lysis In ruminants,in the first duplicationmonths2. Conclusions of pregnancy. of the IFNW In generodents, generated pituitary interferon- prolactinτ (PRL)(IFNT) released secreted in by response the blastocyst to coitus andinhibits acting 20-hydroxystero on the endometriumid dehydrogenase to inhibitIn the the same (20 prostaglandinα way-HSD), that itthis is difficultF2 functionα (PGF2 to define beingα) luteolytic a subseque border between signal.ntly madnessassumed In elephants, and by normality placental the PRL lactogensgene (PL-1 and PL-2). In ruminants, duplicationin the field ofof humanthe IFNW psychiatry, gene generated the definition interferon- of a normalτ (IFNT) ovary is secreted puzzling. by The the norm blastocyst and expressed in the placenta is responsible for pregnancy maintenance by accessory corpora lutea. In red, proteins born after acting on the endometrium to inhibitin mammals the prostaglandin is that the adult F2 ovaryα (PGF2 containsα) luteolytic multiple structural signal. In units elephants, that are follicles, the PRL gene ex- gene duplication (adapted from [58]).where each follicle contains a single oocyte interacting with its surrounding granulosa cells. pressed in the placenta is responsible for pregnancy maintenance by accessory corpora lutea. In red, proteins born after Some of the above-cited examples deviate from the norm, as for example: the existence of gene duplication (adapted fromThis [58]).follicles evolutionary containing multiple incongruity oocytes or is conversely in reality follicles most losing likely their not oocytes due prematurely to the ovary itself, but to the placenta,with granulosa which cells issynthesizing an organ and that secreting has evolvedsteroids in a rather cyclic manner rapidly and/or between forming mammals. It 2. Conovarianclusion tumors.s The norm is also that most of the quiescent primordial follicles forming the is very probableovarian reserve that in the ovarian ovarian cortex corpus will degenerate luteum, throughout in order the to life; ensure the possibility its maintenance to two weeks afterInactivate the ovulation same them after way in decades largethat ofit mammals, quiescenceis difficult by expresses to disrupting define the thea border Hippo receptor signaling between adapted pathway madness to gives the luteotrophicand normality factorsin the expressedthem field an of unexpected human by the opportunity placentapsychiatry, to of develop. thethe speciesdefinitio Concerning inn question.of ovarian a normal endocrinology, ovary ais dogma puzzling. The norm was also developed to suggest that the ovulation rate and the follicular size at ovulation in mammalsare fixed for iseach that animal the adult species ovary and depended contains on a dialoguemultiple existing structural between units ovarian that are follicles, 2. Conclusionswheresteroids each andfollicle inhibin contains secreted by a growingsingle folliclesoocyte and interacting the pituitary with hormones, its surrounding FSH and granulosa cells.In theLH.Some The same subsequentof the way above-cited that discovery it is difficult and examples elucidation to define devia of the a roleste border from that the the between ovarian-derived norm, madness as for factors example: and normality the exist- ence suchof follicles as AMH andcontaining the BMP family multiple play in oocytes regulating or both conversely the number and follicles the size oflosing the their oocytes in the fieldovulating of human follicles are psychiatry, new insights, the which definition significantly of modify a normal the aforementioned ovary is puzzling. dogma. The norm inprematurely mammalsSignificant is with thatquestions thegranulosa still adult remain, ovary cells however. synthesizing contains For example, multiple and why havesecreting structural different steroids animal units species thatin a arecyclic follicles, manner whereand/or eachevolved forming follicle so many containsovarian different tumors. a strategies single oocyteThe to maintain norm interacting their is al corpusso withthat luteum most its during surrounding of gestation?the quiescent granulosa primordial cells. Somefollicles of the forming above-cited the ovarian examples reserve deviate in the from ov thearian norm, cortex as will for example: degenerate the throughout existence of the follicleslife; the containing possibility multiple to activate oocytes them or converselyafter decades follicles of quiescence losing their by oocytes disrupting prematurely the Hippo withsignaling granulosa pathway cells synthesizing gives them an and unexpected secreting steroids opportunity in a cyclic to develop. manner Concerning and/or forming ovarian ovarianendocrinology, tumors. The a dogma norm is was also thatalso mostdeveloped of the quiescentto suggest primordial that the ovulation follicles forming rate and the the ovarianfollicular reserve size in atthe ovulation ovarian are cortex fixed will for degenerate each animal throughout species and the depended life; the possibility on a dialogue to activateexisting them between after decades ovarian of steroids quiescence and byinhibin disrupting secreted the by Hippo growing signaling follicles pathway and the gives pitui- themtary an hormones, unexpected FSH opportunity and LH. The to develop. subsequent Concerning discovery ovarian and elucidation endocrinology, of the a dogmaroles that wasthe also ovarian-derived developed to suggestfactors such that as the AMH ovulation and the rate BMP and family the follicular play in sizeregulating at ovulation both the arenumber fixed for and each the animal size of the species ovulating and depended follicles are on new a dialogue insights, existing which significantly between ovarian modify steroidsthe aforementioned and inhibin secreted dogma.by Significant growing question follicless and still the remain, pituitary however. hormones, For example, FSH and why LH. The subsequent discovery and elucidation of the roles that the ovarian-derived factors such as AMH and the BMP family play in regulating both the number and the size of the Genes 2021, 12, 928 11 of 13

As we can see, the ovary of mammals is capable of functional quirks that can be linked to genetic modifications, to natural intra-species alterations, to differences in inter- species functioning, or to environmental effects. Ovarian function can also be “jostled” by artificially stimulating the recruitment of primordial follicles that one might have thought “lost” for reproduction. These “dysfunctions” are most likely largely related to the fact that the ovary is an organ in perpetual cyclical modification and that it is this level of plasticity that opens the “doors of entry” to functional modifications.

Author Contributions: Conceptualization, P.M.; validation, P.M., K.M. and D.M.; writing—original draft preparation, P.M., K.M. and D.M.; writing—review and editing, P.M., K.M. and D.M. supervi- sion, P.M. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

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