BOR Papers in Press. Published on May 13, 2015 as DOI:10.1095/biolreprod.115.128660

MINIREVIEW The ovarian antral follicle - Living on the edge of hypoxia or not?1 Jeremy G. Thompson,2,3,4,5 Hannah M. Brown,3,4,5 Karen L. Kind,6 and Darryl L. Russell3,4 3 The Robinson Research Institute, The University of Adelaide, Adelaide South Australia, Australia 4 School of Paediatrics and Reproductive Health, The University of Adelaide, Adelaide South Australia, Australia 5 Australian Research Council Centre of Excellence for Nanoscale BioPhotonics, The University of Adelaide, Adelaide South Australia, Australia 6 School of Veterinary and Animal Sciences, The University of Adelaide, Adelaide South Australia, Australia Summary sentence: Hemoglobin may play a role in preventing hypoxia in oocytes within antral follicles, while also facilitating luteinization following the LH surge. Running title: Are antral follicles hypoxic? 1

J.G.T. is supported by a National Health and Medical Research Council Fellowship ID 1077694. 2 Correspondence: Jeremy Thompson, School of Paediatrics and Reproductive Health, Medical School South, Frome Road, Adelaide 5005 SA Australia. E-mail [email protected] ABSTRACT Oocytes within antral follicles are thought to have restricted access to oxygen (O2) as the follicle vascularity is not adjacent and both granulosa and cumulus cells are metabolically active. Indeed, measured follicular antrum pO2 is regarded as low, but accurate and direct measurement represents a technical challenge that has yet to be overcome. The oocyte itself is highly dependent on oxidative phosphorylation for survival and competence for further development following fertilization, and it has been suggested that follicular pO2 levels are correlated with this capacity for further development. It is clear that gonadotrophins are involved in regulating antrum formation, follicle vascularization, cellular differentiation and the Hypoxia Inducible Factors (HIF), which are mainly regulated by dissolved O2 concentration. A newly discovered player in this story is the intracellular production of hemoglobin by both granulosa and cumulus cells, as well as the oocyte. Furthermore, cellular hemoglobin levels are dynamic, responding to the ovulatory luteinizing hormone (LH) surge. We hypothesize that this gas transport and antioxidant molecule is involved in: (a) the prevention of hypoxic-response signaling via HIFs within the pre-ovulatory antral follicle; (b) the transition of granulosa cells to luteal tissue, by facilitating the stabilization of HIFs, enabling the induction of luteinization signaling. Another possible role is via sequestering nitric oxide (NO) during the ovulatory period, which may facilitate the resumption of meiosis in the oocyte. Testing these hypotheses will be challenging, but important if the regulation of ovarian function is to be fully understood. Key words: follicle, oocyte, oxygen, hypoxia, HIF, hypoxia inducible factor, hemoglobin, VEGF, ovary, ovarian vascularization, nitric oxide, NO

Copyright 2015 by The Society for the Study of Reproduction.

INTRODUCTION Certain highly specialized tissues in the body are not directly adjacent to vascular support. For example, within the eye, the cornea is avascular, as a blood supply and capillary network would significantly interfere with its unique function. Another example is the early embryo prior to implantation; the inner cell mass cells as well as the primitive endoderm at the blastocyst stage are surrounded by a sphere of trophectoderm cells, all of which exist in a low O2 environment within the uterus immediately prior to and during early implantation [1-3]. Arguably the most intriguing of avascular tissues are those located within the antral follicle of the ovary. The antral follicle is comprised of multiple layers of different cell types that have highly specialized functions. The outermost cellular layers of the follicle are the theca cells which arise through differentiation of the surrounding stroma from the secondary (multi cellular layers, no antrum) stage. The thecal layers contain the follicular vasculature and steroidogenic cells of the follicle, synthesizing and secreting androgen. A basement membrane separates the theca and granulosa cell layers which excludes all vasculature from the follicle interior. Granulosa cells aromatize androgen to produce estrogen and also produce protein hormones controlling regulation of the female reproductive cycle and oocyte maturation. As follicles grow an antrum, a fluid filled cavity, forms in the granulosa compartment. It is thought that antrum formation is associated with a critical follicle size [4], establishment of an osmotic gradient via proteoglycan secretion from granulosa cells [5] and is also dependent on follicle stimulating hormone (FSH) action [6]. The antrum serves to physically separate the granulosa layers which diverge into 2 distinct subpopulations of cells. The granulosa cells line the follicle wall; the cells surrounding and in intimate contact with the oocyte become the cumulus cells. These have a separate role as the nurse cells for the growing oocyte, separate hormone responsiveness, and a distinct fate [7, 8]. Located acentrically within the follicle is the cumulus-oocyte complex, comprising the cumulus of 8-10 cell layers surrounding the maturing oocyte. Thus the oocyte is separated from the vasculature and hence O2 and nutrients must cross many different cell layers before they become available to the oocyte. The formation of the vascular bed around follicles is regulated, as in most tissues, by VEGFA and other angiogenic factors [9-12]. It is unequivocal that vascularization, hence pO2, are dynamically managed in the developing ovarian follicular environment. Understanding the mechanism and reasons for this is compelling, as it more fully explains the physiological needs of the developing oocyte and the signaling interactions among endocrine factors and environmental conditions that normally control homeostasis in the follicle. In this mini-review, we propose that the dissolved O2 concentration contributes to the dynamic environment of the antral follicle, and is involved with complex signaling between gonadotrophins, hypoxia inducible factors and hemoglobin, all designed to maintain a nonhypoxic environment during pre-ovulatory growth and a transient hypoxic environment enabling post-ovulatory cellular differentiation. IMPORTANCE OF OXYGEN AND OXIDATIVE METABOLISM FOR OOCYTE DEVELOPMENT The oocyte is a unique cell; it is the largest cell in the body when mature; in contrast to somatic cells, it cannot be propagated in culture; it is surrounded by a unique glycoprotein matrix; and it is entirely dependent on oxidative phosphorylation for its energy requirements and therefore its own viability [13, 14]. This latter point is an enigma: the cell that is the focus of female

reproduction develops while purposefully sequestered within an avascular structure, and yet is dependent on a minimal level of dissolved oxygen for survival. Because of the lack of close apposition to a capillary bed, it has been proposed that the dissolved O2 concentration that the oocyte is exposed to in situ may be a determinant of oocyte viability. However, a major deficiency has been the lack of credible data reflecting accurately the dissolved O2 concentration adjacent to an oocyte. Determination of dissolved O2 within antral follicles is fraught with technical difficulties [15]. All data obtained to date has relied on sampling the follicular fluid collected from the follicle antrum by aspiration, then rapidly assessing dissolved O2 content [15]. Nevertheless, in a seminal publication, Van Blerkom described the large antral follicle as a low pO2 environment, and reported that the health of the oocyte was indeed dependent to the pO2 within the follicle, whereby a critical concentration was required for oocyte competence [14]. However, this concept of follicular O2 equating to oocyte developmental competence is controversial; it has been questioned by Huey et al. [16] using human data, arguing that blood flow around the follicle is more significant in determining oocyte health, rather than simply intra-follicular pO2. DO IN VITRO MATURATION STUDIES INFORM THE OPTIMAL INTRAFOLLICULAR OXYGEN CONTENT FOR OOCYTES? It is perceived that cumulus-oocytes complexes (COC) matured in culture benefit from a low pO2, which according to Henry’s law, allows direct comparison of dissolved O2 levels, so long as the solubility of O2 does not alter between different fluids. However, studies examining different pO2 levels for in vitro oocyte maturation have not been useful in determining the optimal dissolved O2 content, as there has been both a limited range of pO2 levels assessed (either low, around 5-7% of gas composition, versus atmospheric ≈ 20%, depending on altitude) and a lack of consistency in results following maturation under these dissolved O2 levels [17]. Differences in origin of species of oocyte, medium composition, macromolecule and protein supplementation and density of COC culture are examples of variables that appear to interact with the impact of different pO2 levels under in vitro maturation conditions [17-23]. Perhaps more relevant is oxygen uptake data by COCs comparing competent and less-competent oocytes. Oxygen consumption and ATP production is associated with improved developmental capacity of in vitro matured mouse COCs [24]. However, these more competent in vitro derived COCs differed significantly in their metabolic profile compared to in vivo derived COCs, which exhibit much more efficient mitochondrial activity for ATP production [24]. Therefore, it doesn’t necessarily follow that more O2 consumption is related to greater oocyte competence. MODELLING THE FOLLICULAR ENVIRONMENT Since direct measurement of intra-follicular pO2 is technically difficult, an alternative approach has been to mathematically model the theoretical content, based on more accurately measurable ovarian arterial and venous O2 levels, and tissue respiration rates. Early attempts to do this relied on inaccurate assumptions regarding O2 diffusion rates in tissues [25], which quite erroneously (as discussed by Clark et al. [26]) predicted that the oocyte was exposed to an anoxic environment. Elegant modelling by Redding and colleagues [4, 27, 28] and Clark and Stokes [29] suggests that the formation of the follicular antrum improves gas and substrate diffusion into a spherical tissue, in which cellular proliferation is required for growth. By increasing surface area substantially, while minimizing the number of cell layers, cell proliferation can still occur and follicles are able to grow in size without nutrient deficiency at the core of a sphere. This

seems, at least, one strategy for tissues, including the ovarian antral follicle, to enable adequate gas and nutrient diffusion to occur when size and metabolic activity would constrain diffusion across cell layers. In an analogous situation, this strategy was also predicted by Leese to explain the formation of the blastocoel cavity at the blastocyst stage in embryos [30]. Nevertheless, despite antrum formation, Redding and colleagues calculated that oocytes in human antral follicles exist in a relatively low pO2 environment, with levels varying between 11-51 mmHg, the variability being attributed to the number of layers of mural granulosa cells which consume and impede diffusion of O2 to the follicular antrum [28]. Such low levels of O2 can elicit a hypoxic response, therefore we hypothesize that Hypoxia Inducible Factor (HIF)-mediated gene expression would be required to support the health of the follicle (and therefore the oocyte) in the face of low and possibly variable O2 levels. HIFs IN THE OVARIAN FOLLICLE Hypoxia Inducible Factors (HIFs) are highly conserved, heterodimeric transcription factors comprising an α and β subunit, and are a member of the basic helix-loop-helix (bHLH)-PAS domain family of transcription factors. The α subunit contains the oxygen-sensitive domains and both subunits are ubiquitously expressed, although mRNA for the α subunit is regulated by many factors, such as growth factors, hormones and pO2. HIF activity is highly regulated by post-translational modifications as a result of O2 concentration; in the presence of O2, nuclear translocation is unable to occur and the α subunit is rapidly ubiquinated. Both these processes are regulated by hydroxylase enzymes, which catalyze hydroxylation of the HIF proteins. As a consequence, both HIF1 and 2 proteins have very short half-lives under normal cellular oxygenation, but HIF is stabilized under low oxygen activating the “hypoxic stress response” signaling pathway. Rather surprisingly, given the potential for low oxygen content within antral follicles, there is a paucity of evidence that HIF is involved in the development of the mammalian ovarian antral follicle. Using various transfection reporter constructs in a primary rat granulosa cell culture model, Alam and colleagues [31, 32] determined that FSH regulation of granulosa cell gene expression involved HIF-signaling, mediated through the PI3K/Akt pathway. They concluded that follicle stimulating hormone (FSH) stimulated HIF1α protein translation in conjunction with an increase in HIF1 activity through increased downstream gene activation, independent of the enhanced HIF stabilization. These experiments utilized culture under atmospheric oxygen conditions but HIF activity was artificially stabilized by either a proteasome inhibitor or hydroxylase inhibitor (CoCl2), both of which are common approaches to mimic a low O2 environment in culture.. Nevertheless, they also concluded that unravelling how FSH regulates HIF activity (not just protein stabilization) under normoxic culture conditions will be “complex and requires input from multiple signaling pathways” [32]. This has been recently echoed by Rico [33] and Meidan [34]. Using mouse granulosa cells transfected with a mutated HIF response element (HRE) for the Vegfa gene promoter, Rico and colleagues demonstrated that FSH-induced (and indeed, luteinizing hormone (LH)-induced) Vegfa expression in vitro was nullified in these cells. This clearly supports an FSH-mediated, HIF dependent mechanism for VEGFA production, but then queried how FSH could do so [33]. In a recent publication, Yalu and colleagues [35] demonstrated that CoCl2 was absolutely required for cAMP-mediated HIF1α protein stabilization in bovine granulosa cell cultures. What is consistently observed in all these studies is FSH (and LH) stimulation of HIF actions occurs only in conjunction with

hypoxic conditions; however what remains unclear is the role for low O2 in mediating the actions of gonadotrophins in the ovarian follicle in situ. In contrast to the subtle HIF activation with FSH, several laboratories [11, 33, 35-39] have shown that HIF activity is greatly up-regulated following the ovulatory signal and is significantly involved in the differentiation of the mammalian follicle into the corpus luteum. We developed a HIF activation-reporter transgenic mouse [37], and found that following ovulation induction, differentiating and ovulating antral follicles, as well as established corpora lutea, have significant levels of reporter activity [37]. In contrast, all pre-ovulatory follicles, regardless of developmental stage (including all non-ovulatory antral follicles) revealed no HIF reporter activity within the mural granulosa, cumulus layers or oocyte (Figure 1). This suggests no obvious role for HIF activity within the growing antral follicle. We have subsequently expanded these observations by demonstrating that COCs, collected prior to the ovulatory surge, reveal no evidence for HIF activation (determined by HIF1α western analysis and transgenic HIF-reporter activity) [40]. Such observations are at odds with the mathematical modelling, which suggest the pO2 of antral follicles should be considered close to, if not hypoxia , where HIF-mediated response to low oxygen levels, along with FSH stimulation, are predicted to be required to sustain healthy follicle growth and oocyte development. In contrast, in vitro maturation of follicular COCs under culture conditions that mimic the inferred in vivo low- to very low- (5% and 2%) follicular O2 levels revealed significant increases in HIF1α protein and HIF-reporter activity [40]. Likewise, physiologically low O2 following an ovulatory stimulus appears essential in order for HIF protein stability, and therefore increased HIF activity. This raises the question of whether there is a mechanism operating within ovarian antral follicles prior to ovulation which prevents hypoxic-induction of HIF to occur, that is then rapidly lost at ovulation or during in vitro maturation. HEMOGLOBIN WITHIN THE OVARIAN FOLLICLE In order to better understand the interpretation of these in vivo and in vitro experiments we undertook microarray analysis of mouse COCs matured under in vivo vs. in vitro conditions [41]. We discovered that hemoglobin mRNAs (Hba-a1 and Hbb) were detected within in vivo, but not in vitro matured cumulus cells of the ovarian antral follicle [41]. To rule out suspected blood contamination during collection and tissue processing, we found very low to undetectable transcript abundance for erythrocyte gene markers (Gata1, Ahsp and Slc4a1). Furthermore, we determined that follicular transcripts for Hba-a1 and Hbb are hormonally regulated, being upregulated around the time of ovulation (12 h post hCG), then declining to pre-hCG administration levels [42]. We have also found transcripts for heme biosynthesis, which is unsurprising given the necessity of heme for other follicular enzymes, such as the cytochromes, but supports the possibility that active O2-binding hemoglobin may be assembled. Furthermore, peri-ovulatory human granulosa and cumulus cells had similarly high HBA1 and HBB transcript levels, but not of GATA1 [42]. Other non-hematopoietic tissues are also known to express hemoglobin, such as several neural cell types [43], hepatocytes [44], mesangial cells [45] macrophages [46], the eye’s cornea [47] and retina [48]. Arterial endothelial cells produce the α-chain of hemoglobin, which plays a significant role in nitric oxide (NO) regulation [49]. Some of these tissues appear to lack heme, therefore have no O2 binding capacity, suggesting functions other than O2 transport. However, other examples seemingly correlate with a need for O2 (for example [47, 50]).

Adult hemoglobin has a quaternary structure, comprising two α and two β globulin chains. Within each of these chains a porphyrin ring surrounds an Fe2+ ion, the heme group. There are several other forms of hemoglobin, which include a fetal form (substitute’s two β globulins with two γ globulins) and genetic variants leading to diseases such as sickle cell anaemia and thalassemia, while in recent years, six additional globins have been described in vertebrates (androglobin, cytoglobin, globin E, globin X, globin Y and neuroglobin) [51]. Hemoglobin has other functions: as an antioxidant and iron scavenger; and binding and transport of other gases, such as NO, CO and CO2. The oxygen carrying capacity of hemoglobin is also regulated, involving the competitive binding to heme of 2,3-bisphosphoglycerate (2,3-BPG) and O2. Significantly, ovarian granulosa cell tumours have greatly up-regulated RNA expression of the 2,3-bisphosphoglycerate mutase enzyme, which produces 2,3-BPG [52]. This would substantially reduce the O2 binding capacity of hemoglobin in these tumour cells relative to nonmalignant granulosa cells, such that O2 would readily dissociate from hemoglobin. WHAT ARE THE POSSIBLE ROLES OF HEMOGLOBIN IN THE OVARIAN FOLLICLE? Could hemoglobin play a role in providing the oocyte with sufficient O2 to enable efficient oxidative phosphorylation and avoid hypoxic stress responses? Does hemoglobin buffer against dynamic changes in pO2 during antral follicle growth? Is this why there appears to be no HIF signaling in the in vivo derived cumulus-oocyte complex prior to the ovulatory surge, despite theoretical low pO2 levels for this to occur? If hemoglobin was to have this function, hemoglobin-O2 binding would require a functional heme group; this has yet to be verified for granulosa or COC-localized hemoglobin. Nevertheless, it is well known that heme is required for steroidogenesis, therefore likely to be present in both these cell types. Validation of this hypothesis is problematic, as a targeted knockout approach of either Hba-a1 or Hbb within the antral follicle is deemed intractable for conditional targeting design by the international knockout mouse consortium [53]. Targeted CRISPR-Cas9 approaches are being rapidly developed and may yet offer a resolution to this problem. The dynamic nature of both hemoglobin and other related gene expression during the periovulatory period within cumulus and granulosa cells points to a probable role in the ovulatory process, in particular, the transition from granulosa cell to luteal cell phenotype. It has been reported that the pO2 of the follicle decreases during the peri-ovulatory period [54]. This coincides with an increase in follicular hemoglobin mRNA and protein levels, concurrently with a decrease in the abundance of the bisphosphoglycerate mutase mRNA levels, the enzyme that regulates 2,3-BPG levels [42]. The interaction between hemoglobin-bound O2 and allosteric effector 2,3-BPG is a very well characterized functional modification. Briefly, 2,3-BPG displaces O2 from hemoglobin, thus increasing the dissolved O2 availability in respiring tissues as a consequence of O2 demand and subsequent dissolved O2 gradient. A reduction in levels of 2,3-BPG causes a ‘tighter’ association of O2 to hemoglobin, shifting the hemoglobin-O2 dissociation curve further to the left, requiring a lower level of tissue pO2 for dissociation to occur. It is possible that O2 is increasingly bound to hemoglobin, as hemoglobin levels rapidly rise following the LH surge, while simultaneously there is a decrease in intracellular 2,3-BPG levels, causing a transient hypoxic state. The importance of this may relate to the rapid upregulation of Hypoxia Inducible Factor (HIF) activity within granulosa cells during this transition. We have shown that prior to the ovulatory surge, little HIF activity is observed in antral follicles [37, 40]. However, following the LH surge, HIF activity is significantly increased

and is a key player in the earliest signaling of luteinization [36, 37]. Although there are certainly adjunct mechanisms involved in this cellular differentiation, it is conceivable that granulosa cell intracellular hemoglobin during luteinization sequesters free O2, promoting a reduced intracellular O2 environment, thus increasing HIF stability, which in turn actively mediates signaling that drives luteinization (Figure 2). An alternate explanation involves the potential role of hemoglobin as an antioxidant molecule. LH induction of ovulation in rats is accompanied by an increase in reactive oxygen species (ROS) generation that facilitates epidermal growth factor receptor (EGFR) and downstream signaling events [55]. At the same time, intracellular antioxidant mechanisms increase in response to this activity [56]. Interestingly, Yalu et al [35] found that H2O2 treatment of bovine granulosa cells stabilized HIF1A protein. Hemoglobin is a known antioxidant [45] and its increased expression over the peri-ovulatory period may assist protecting follicular cells, especially the oocyte, from increased ROS levels over this period. Hemoglobin also sequesters other gases, of which nitric oxide (NO) is one of considerable interest. An alternative or additional function for hemoglobin could involve NO regulation during the peri-ovulatory period. A regulatory role for NO in ovulation is supported in most species, with sources thought to be both inducible Nitric Oxide Synthase (iNOS) and endothelial NOS (eNOS), with iNOS activity increasing with the LH surge [57-62]. However, this appears to be at odds with a hypothesis that increased hemoglobin activity, initiated by the LH surge, would be involved in sequestering NO activity. An alternative theory is a role in diminishing soluble guanylate cyclase (sGC) activity. NO is a known sGC activator [63, 64] and cGMP is a fundamental component of oocyte meiosis arrest [65, 66]. Potentially, hemoglobin-NO binding by an increase in hemoglobin levels following hCG may reduce NO-induced sGC activity. Nevertheless, although attractive, sGC activity is not associated with a regulatory role for cGMP production in granulosa cells of large antral follicles [67]. Rather, the naturetic peptides, NPPC (C-type natriuretic peptide) and its putative receptor, natriuretic peptide receptor 2 (NPR2), a membrane-bound guanylate cyclase, are the regulators of meiosis, which are impacted by the EGF-like peptides following the LH surge [68, 69]. CONCLUSION The ovarian antral follicle has a high proliferative rate and metabolic activity; while the oocyte seemingly has O2 sensitivity. This presents an intriguing set of physiological challenges for the developing follicle that remain to be fully understood. The prevailing evidence suggests that a low O2, but not hypoxic, environment exists in the follicle, most likely for the protection of the oocyte from possible oxidative damage while delivering sufficient O2 to meet its oxidative phosphorylation demands. Meanwhile, the metabolically active granulosa and cumulus cells tolerate the lower O2 environment without the need to activate a hypoxic stress response, yet the HIF/hypoxia pathway is an important component of the hormone responsive gene regulation in follicular somatic cells. Consistent findings that FSH and LH induce VEGFA, which is facilitated by a HIF-dependent mechanism under low pO2, indicate complex regulation of follicle vascularization via female endocrine signaling. The follicular antrum itself is one strategy to satisfy O2 and nutrient supply to the avascular compartment of the follicle. Importantly, the discovery of regulated hemoglobin and related gene expression in granulosa and cumulus cells potentially further explains these cells capacity for tolerating a low dissolved O2 concentration, and possibly changes in NO, through a localized intra-follicular gas supply regulating mechanism.

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FIGURE LEGENDS Figure 1. A pre-ovulatory follicle (left) adjacent to a mature corpus luteum (right) obtained from an ovary of a C57BL/6-Tg(HRE(4)-SV40-EGFP transgenic mouse. Note the green fluorescence from the HIF-responsive GFP-reporter construct in the corpus luteum and the lack of GFP expression in the follicle in tissues enclosed within the basement membrane. Nuclei of cells stained with DAPI. Bar = 50 μm. Figure 2. A hypothesis for the roles of O2, HIF and hemoglobin during the peri-ovulatory period. Prior to the ovulatory signal (LH; hCG), hemoglobin and HIF levels are low and follicular oxygen concentration is low, but not hypoxic. Following the LH surge, despite the increased vascular support and vascular permeability to the follicle, dissolved O2 falls, hemoglobin (HBA1, HBB) within the follicle increases, and 2, 3-bisphosphoglycerate decreases (decreased activity of 2, 3-bisphosphoglycerate mutase, BPGM), causing a higher binding affinity for O2 by hemoglobin, decreasing the granulosa/cumulus cell intracellular levels of follicular oxygen, thus triggering an increase in HIF-mediated VEGF induction. Taken from Brown et al. [42] with permission.

Figure 1

oocyte

follicle

corpus luteum

Figure 2