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`EXHIBIT (V
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`DATE 3-;≥.q-
`KRAMM COURTREPOR TING
`
`Endocrinology 149(7):3313-3320
`Copyright C 2008 by The Endocrine Society
`doi: 10.1210/en.2007.1849
`
`Inhibition of Vascular Endothelial Growth Factor in the
`Primate Ovary Up-Regulates Hypoxia-Inducible
`Factor-la in the Follicle and Corpus Luteum
`
`W. Colin Duncan, Sander van den Driesche, and Hamish M. Fraser
`
`Division of Reproductive and Developmental Sciences (W. C.D., S. v.d.D.), University of Edinburgh EH16 4SB, United
`Kingdom; and Medical Research Council Human Reproductive Sciences Unit (H.M.F.), Queens Medical Research Institute,
`Edinburgh EH16 4TJ, United Kingdom
`
`Vascular endothelial growth factor (VEGF)-dependent angio-
`genesis is crucial for follicular growth, and corpus luteum
`formation and function, in the primate ovary. In the ovary
`VEGF can be hormonally regulated, but in other systems, the
`main regulator of VEGF expression is hypoxia. We hypothe-
`sized that hypoxia was involved in the regulation of angio-
`genesis in the cycling ovary. We therefore used immunohis-
`tochemistry to localize hypoxia-inducible factor (HIF)-la in
`the marmoset ovary across the ovarian cycle. We also inves-
`tigated the effect of VEGF inhibition, using VEGF Trap
`(affibercept), on REF-la localization during the follicular and
`luteal phases of the cycle. Finally, we studied the effect of
`chorionic gonadotropin stimulation of the corpus luteum dur-
`ing early pregnancy. Nuclear HIF-la staining was largely ab-
`sent from normally growing preanfral and antral follicles.
`However, there was marked up-regulation of nuclear HIP-la
`
`in the granulosa cells at ovulation that persisted into the
`early corpus luteum. Mature corpora lutea and those col-
`lected during early pregnancy had minimal nuclear HIP-la
`staining. The inhibition of VEGF in the mid-luteal stage
`resulted in a time-dependent up-regulation of luteal nu-
`clear HIP-la staining (P < 0.05). There was never any nu-
`clear REF-la in the theca cells of the follicle, but VEGF Trap
`treatment during the follicular (P < 0.001) or luteal (P <
`0.001) phase increased the proportion of antral follicles
`with nuclear REF-la staining in the granulosa cells. These
`results indicate that HIP-la is up-regulated after vascular
`inhibition, using VEGF Trap, in the follicle and corpus lu-
`teum. However, it is also acutely up-regulated during ovu-
`lation. This suggests a role for HIP-la in both hypoxic and
`hormonal regulation of ovarian VEGF expression in vivo.
`(Endocrinology 149: 3313-3320, 2008)
`
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`THE MAJOR SITE of physiological angiogenesis in
`
`the adult is the female reproductive tract, notably the
`ovary (1). The granulosa cells of the dominant follicle are
`avascular, and yet each granulosa-lutein cell of the mature
`corpus luteum is adjacent to an endothelial cell. Indeed en-
`dothelial cells are abundant in the corpus luteum, accounting
`for approximately 50% of luteal cells, and the corpus luteum
`has a blood supply around eight times, per unit mass, that
`of the kidney (I). We have shown that vascular endothelial
`growth factor (VEGF) is the major regulator of luteal angio-
`genesis in the primate ovary (2). When VEGF action is in-
`hibited, luteal formation continues, but the resulting, poorly
`functioning, corpus luteum has a rudimentary vascular bed
`(3). We have also shown that VEGF is required for ongoing
`function and maintenance of the vasculature of the mature
`corpus luteum (4, 5).
`In addition to formation of the corpus luteum, angiogen-
`esis is required for antral follicular growth. Inhibition of
`VEGF in the follicular phase inhibits follicular growth and
`results in small, poorly vascularized antral follicles, and no
`large or dominant follicles develop (2, 6). Gonadotropins
`
`First Published Online April 3, 2008
`Abbreviations: hCG, Human chorionic gonadotropin; HIF, hypoxia-
`inducible factor; NGS, normal goat serum; PBS-T, PBS plus Tween 20;
`PG, prostaglandin; VEGF, vascular endothelial growth factor.
`Endocrinology is published monthly by The Endocrine Society (httpi/
`wwwendo-sociely.org), the foremost professional society serving the
`endocrine community.
`
`C,
`CD
`
`to
`
`have a clear role in the regulation of follicular growth and
`angiogenesis. Treatment with GnRH antagonists in the fol-
`licular phase also results in small, poorly vascularized, antral
`follicles (7). In addition, GriRH antagonist treatment in the
`luteal phase results in luteolysis and associated vascular
`regression (8).
`It is therefore likely that VEGF expression is regulated by
`gonadotropins in the ovary. This is supported by gonadotropin Cr
`up-regulation of VEGF in primary cultures of luteinized gran-
`ulosa cells (9,10) and by the marked increase in follicular fluid
`VEGF concentrations after the LH surge (11). In addition, the
`temporal changes in VEGF expression across the luteal phase,
`and its up-regulation by human chorionic gonadotropin (hCG)
`in simulated early pregnancy in women (12), also support this
`notion. However in other tissue systems the primary regulator
`of VEGF expression is hypoxia (13). Indeed, it has been reported
`that hypoxia, rather than gonadotropins, is the main regulator
`of VEGF secretion in primary cultures of luteal cells from non-
`human primates (14) and women (15).
`The role of gonadotropins and hypoxia in the regulation
`of ovarian VEGF expression therefore is still not clear. Where
`VEGF is regulated by hypoxia, there is an up-regulation of
`specific transcription factors, notably hypoxia inducible fac-
`tor (HIF)-la that is translocated from the cytoplasm to the
`nucleus (16,17). We hypothesized that HIF-la was involved
`in physiological angiogenesis in the ovary and that its lo-
`calization would change in different functional phases of the
`ovarian cycle.
`
`3313
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`3314 Endocrinology, July 2008, 149(7):3313-3320
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`Duncan et at. • HIF-la in Primate Ovary
`
`Collection of ovaries
`
`Animals were sedated and humanely killed as described previously
`(5, 6). In the luteal phase, the corpora lutea were identified macroscop-
`ically and ovaries bisected along the line of maximal luteal area before
`fixation in 4% (vol/vol) neutral buffered formalin (Van Waters and
`Rogers International Ltd., Leicestershire, UK). After 24 h, the ovaries
`were transferred into 70% (vol/vol) ethanol, dehydrated, and embedded
`in paraffin according to standard procedures.
`
`Immunohistochemistry
`
`To facilitate classification of follicular health, cell death was deter-
`mined by immunohistochemistry for caspase-3 as described previously
`(5). A specific rabbit polyclonal antibody was used for the immunolo-
`calization of FIIF-la (clone H-206; Santa Cruz Biotechnology, Santa
`Cruz, CA) using 5-pm paraffin tissue sections of marmoset ovaries
`prepared on poly-L-lysine-coated microscope slides. These sections were
`dewaxed, rehydrated, washed in PBS, subjected to antigen retrieval by
`boiling in a pressure cooker in 0.01 mol/11ter 1 citric acid (pH 6.0) for
`5 mm, and left to cool to room temperature. All sections were washed
`and placed in 3% (vol/vol) H202/methanol for 30 min, followed by an
`avidin and biotin block (Vector Laboratories, Peterborough, UK) and a
`further block using normal goat serum (NGS; Diagnostics Scotland,
`Edinburgh, UK) diluted 1:5 in PBS containing 5% (wt/vol) BSA
`(NGS/PBS/BSA) for I h at room temperature. Cettions were incubated
`overnight in primary antibody diluted 1:100 in NGS/PBS/BSA at 4 C.
`All sections were then washed twice for 5 min in PBS plus 0.01%
`(vol/vol) Tween 20 (PBS-T; Sigma-Aldrich, Poole, UK) before incubation
`with biotinylated goat antirabbit secondary antibody (Dako Corp., Cam-
`bridge, UK), diluted 1:500 in NGS/PBS/BSA. Incubation lasted for I h
`and was followed by two washes in PBS-I for 5 min. Thereafter sections
`were incubated in avidin-biotin complex-horseradish peroxidase (Vec-
`tor Laboratories) for 1 h according to the manufacturer's instructions. All
`sections were washed in PBS-I (2 X 5 mm) and bound antibodies
`visualized by incubation with liquid 3,3'-diaminobenzidine tetra-
`hydrochloride (Dako). Sections were counterstained lightly with hema-
`toxylin to enable cell identification. Negative controls for each antibody
`examined were performed identically to the above protocol with the
`primary antibody omitted or replaced with nonspecific Igs (Santa Cruz
`Biotechnology). Images were captured using an Provis microscope
`(Olympus Corp. Optical Co., London, UK) equipped with a DCS33O
`camera (Eastman Kodak Co., Rochester, NY), stored on a computer
`(Hewlett-Packard, Portland, OR), and assembled using Photoshop 7.0.1
`(Adobe Systems Inc., Mountain View, CA).
`
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`
`Analysis of sections
`
`Sections were analyzed by an observer blinded to treatment type. For
`the analysis of corpora lutea, the percentage of luteal steroidogenic cells
`with clear nuclear HIP-la immunostaining was recorded using a strat-
`ified random sampling technique (24). Both ovaries were examined for
`each animal, but where there was more than one corpus luteum, the
`score was averaged for that animal before statistical analysis. For the
`analysis of follicular granulosa cell HIP-la immunostaining, all antral
`follicles (<1 mm in size) (25) in midline sections of both ovaries were
`counted and classified as positive where there was marked nuclear
`HIF-la staining in the all the granulosa cells, negative where no dear
`nuclear immunostaining could be detected in any granulosa cells, or
`intermediate where there was patchy or faint nuclear HIF-la staining in
`the granulosa cells.
`
`Statistical analysis
`
`The percentage of nuclear HJF-la: stained steroidogenic cells in cor-
`pora lutea was analyzed using the Kruskal-Wallis test. The proportion
`of immunostained antral follicles, in the follicular and luteal phases after
`treatments, was analyzed using a 2 test. Statistical differences where
`P <0.05 were considered significant.
`
`We therefore investigated the localization of HIP-la protein
`in the primate ovary during the follicular and luteal phases of
`natural cycles. We also studied the effect of cycle manipulation
`on F{IF-la localization using three strategies: 1) inhibition of
`VEGF in the follicular phase, that is known to up-regulate
`follicular VEGF expression (6), 2) inhibition of VEGF in the
`midluteal phase that has been shown to inhibit endothelial cell
`survival and vascular integrity (5), and 3) gonadotrophic stim-
`ulation of the corpus luteum in early pregnancy that in women
`causes an up-regulation of VEGF (12).
`
`Materials and Methods
`
`Animals
`
`Adult female common marmoset monkeys (Callit hrix jacchus) with a
`body weight of approximately 350 g and regular ovulatory cycles (28 d
`cycle length) with ovulation on d 8 were housed together with a younger
`sister or prepubertal female as described previously (6). Blood samples
`were collected three times per week by femoral venipuncture without
`anesthesia, and plasma was subjected to progesterone assay for the
`presence of ovulatory rises, as described previously (5).
`
`Treatment
`
`Experiments were carried out in accordance with the Animals (Sci-
`entific Procedures) Act, 1986, and were approved by the local ethical
`review process committee. In the first set of experiments, the follicular
`phase was investigated. To synchronize timing of ovulation, during the
`pretreatment cycle, marmosets were given I jLg prostaglandin (PG) F2,,
`analog (cloprostenol, Planate; Coopers Animal Health Ltd., Crewe, UK)
`im in the mid- to late luteal phase (d 13-15) to induce luteolysis (6). This
`method of synchronizing follicular recruitment is followed by follicle
`selection on cycle d 5 and ovulation between d 9 and 11 (18) in which
`the day of synchronization is d 0.
`To block VEGF, we used VEGF Trap (aflibercept), a recombinant
`chimeric protein comprising portions of the extracellular domains of the
`human VEGF receptors 1 and 2 expressed in sequence with the Fc
`portion of human Ig (19). VEGF Trap binds all isoforms of VEGF-A,
`VEGF-B, and placental growth factor. Four marmosets were treated with
`VEGF Trap at a dose of 25 mg/kg' injected sc at the time of PGF2,,
`treatment (d 0), and four were treated on d 5 of the cycle (midfollicular
`phase). Control animals (n = 4) were treated with human Fc (25 mg/
`kg' Sc). Ovaries were collected on d 10. These ovaries had been used
`in previous studies to assess the effect of VEGF Trap on the follicle (20,
`21). Ovaries from untreated marmosets on d 5 (n = 4) were also available
`(20).
`In the second set of experiments, luteal phase administration of VEGF
`Trap was used. In these animals ovulation was designated luteal d 0 and
`defined as the day preceding a rise from follicular phase (<32 runoll
`liter') progesterone followed by a progressive increase in progesterone
`(5). These criteria have been used in our and other colonies to accurately
`identify the day of ovulation to within ± 1 d (22). Marmosets exhibiting
`at least one ovulatory cycle immediately before being recruited into the
`study were selected. Marmosets were treated with a single injection of
`25 mg/kg-1 sc VEGF Trap in the midluteal phase (d 8-10). Control
`marmosets were treated with recombinant human Fc (25 mg/kg' Sc).
`Ovaries were collected 1, 2, and 4 d (n = 4 per group) later (5). Control
`animal ovaries (n = 4 per group) were collected 2, 4, and 8 d later. These
`ovaries have been investigated in a previous study assessing inhibition
`of luteal VEGF in the postangiogenic window (5).
`In a third set of experiments, ovaries from pregnant marmosets were
`investigated. These ovaries have been collected and studied as part of
`a previous investigation into the rescued corpus luteum of the marmoset
`(23). At the time of PGF,, administration, a fertile male was introduced,
`and marmosets were killed 28 d after ovulation and confirmed pregnant
`by the presence of trophoblast in serial sections of the uterus and plasma
`levels of chorionic gonadotropin of greater than 20 ng/m1' (23).
`
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`Duncan et at. • [HF-in in Primate Ovary
`
`Endocrinology, July 2008, 149(7):3313-3320 3315
`
`diiq wojj papeojuMog
`
`/academic.oup.coni/endo1articIe/14917I3313/245434 by guest on 22 March 202 2
`
`FIG. 1. Immunolocalization of HIF-la
`in the marmoset follicle. A, Preantral
`follicle in the marmoset ovary with
`neighboring primary (F) and anti-al
`follicle (A). B, Antral follicle in differ-
`ent ovary showing light cytoplasmic
`HIF-la staining in various follicular
`cell types. C, Atretic follicle showing
`specific patchy nuclear staining for
`HIF-la (arrows). D, Follicular phase
`ovary with selected anti-al (A) follicle
`with no staining with a neighboring
`atretic follicle (AF) demonstrating clear
`nuclear HIF-la immunostaining in the
`granulosa cells layer (arrows). E, Pre-
`ovulatory dominant follicle showing no
`nuclear HIT-la immunostaining. F,
`Luteinizing follicle during ovulation
`demonstrating intense nuclear HIT-la
`staining in the granulosa cells (arrows).
`0, Oocyte; 0, granulosa cells layer; T,
`theca cell layer; A, antral cavity. Scale
`bar, 100 Am.
`
`.6
`
`'0
`
`T.
`
`C
`
`F
`
`A
`
`Results
`HIP-1a localization in the follicle
`
`In the normal follicular phase, there was light HIP-la
`immunostaining in the cytoplasm of cells of the developing
`follicle, including the oocyte, theca cells, and granulosa cells
`(Fig. I). There was no clear nuclear HIF-la localization in
`preantral (Fig. 1A), antral (Fig. 13), selected (Fig. 1D), or
`dominant follicles (Fig. 1E) in all ovaries and follicles exam-
`ined. However, in atretic follicles, nuclear HIF-la immune-
`staining could clearly be detected in the granulosa cell layer
`(Fig. 1, C and D) in all tissue sections examined. Although
`there was no nuclear localization of HIP-la in the preovu-
`latory follicle (Fig. 1E), ovulation was associated with intense
`nuclear HIF-la staining in all granulosa cells (Fig. IF).
`
`The effect of VEGF inhibition on follicular HIP-ice
`localization
`
`After exposure to VEGF Trap in the follicular phase, the
`light cytoplasmic staining of the oocyte and primordial, pri-
`mary (Fig. 2A), and secondary (Pig. 23) follicles seen in
`control ovaries was maintained. Nuclear immunostaining
`could not be detected in any follicles of these stages. No
`staining could be detected in negative control sections
`(Fig. 2, C and I). However, in larger preantral follicles, nu-
`clear HIF-la expression could now be detected in some gran-
`ulosa cells (Fig. 2D). However, the most marked change was
`the appearance of antral follicles with marked nuclear stain-
`ing in the granulosa cell layer (Fig. 2E). In contrast, antral
`follicles from control ovaries showed minimal nuclear
`HIF-la localization (Fig. 217).
`Where nuclear HIF-la immunostaining was seen, there
`were two apparent patterns: intense staining in all granulosa
`cells (Fig. 2, E and C) or light (Fig. 2G), patchy (Fig. 2H)
`staining in the granulosa cell layer. Because atresia is asso-
`ciated with increase HIF-la immunostaining, atretic folli-
`cles were identified by immunostaining serial sections for
`
`caspase-3 (Fig. 2, j and K) and excluded from analysis. The
`effect of VEGF Trap treatment on the proportion of mor-
`phologically healthy antral follicles, with no or minimal
`(Fig. 2L) caspase-3 staining, showing intense (+), light patchy (±)
`or no staining (-) was therefore assessed (Fig. 3A). Inhibition
`of VEGF in the follicular phase increased the proportion of
`follicles with nuclear HIP-la staining in the granulosa cells
`(P C 0.001). Treatment with Trap from d 5 to 10 of the
`follicular phase was associated with more marked nuclear
`staining than treatment from d 0-10 of the follicular phase
`(P <0.05) (Fig. 3A). There was also an effect of Trap treatment
`during the luteal phase on follicular nuclear HIP-la immu-
`nostaining (Fig. 3B). The proportion of antral follicles with
`nuclear HIF-la staining increased with duration of Trap
`treatment (P C 0.001) (Fig. 33). Although there was clear
`nuclear staining in the granulosa cells of follicles after Trap
`treatment, there was never any clear nuclear staining of the
`theca cells of these follicles (Fig. 2, C and H). Like the atretic
`follicles of control ovaries, atretic follicles after VEGF Trap
`treatment showed patchy nuclear HIP-la immunostaining in
`granulosa cells.
`
`HIP-la localization during luteal formation and regression
`
`There was intense nuclear HIF-la immunostaining in the
`luteinizing granulosa cells, but not the theca cells, in the
`periovulatory period (Fig. 4A). As the follicle collapses and
`the corpus luteum begins to form, nuclear HIF-la immuno-
`staining in the granulosa cells is maintained (Fig. 43). How-
`ever, nuclear staining was minimal in every fully formed
`corpora lutea examined. In the natural cycle, there was only
`light cytoplasmic staining of the steroidogenic cells (Fig. 4C)
`of corpora lutes from the mid and late-luteal phases. During
`natural luteolysis, cytoplasmic HIP-la staining of the steroi-
`dogenic cells was evident (Fig. 4D). Although in some sec-
`tions of regressing corpora lutes, there was the occasional cell
`demonstrating possible nuclear localization of HIP-la, these
`
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`3316 Endocrinology, July 2008, 149(7):3313-3320
`
`Duncan it at. • HIF-la in Primate Ovary
`
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`FIG. 2. Immunolocalization of HIF-la
`in marmoset follicles after exposure to
`VEGF Trap. A, Primordial and primary
`follicle in marmoset ovary after expo-
`sure to VEGF Trap from do to 10 of the
`cycle. B, Small secondary follicle after
`the same follicular phase VEGF Trap
`treatment showing no nuclear HIF-la
`staining. C, Serial negative control sec-
`tion of B. D, Larger preantral follicle
`after Trap exposure from d 5 to 10 show-
`ing some nuclear HIF-la staining
`(arrows). B, Antral follicle, with high-
`power detail, in another animal after
`VEGF Trap treatment from d 5 to 10 of
`the cycle showing uniform nuclear im-
`munostaining (arrows) (+). F, Antral
`follicle in follicular phase control ovary,
`with high-power detail, showing no nu-
`clear HIF-la localization (-). 0, High-
`power detail of follicles after 4 d of
`VEGF Trap treatment in the luteal
`phase showing marked nuclear stain-
`ingin granulosa cells (arrows) of follicle
`1 (+) and lighter, more patchy, staining
`(±) in neighboring follicle 2, but not in
`theca cells or either follicle. H, Follicle
`after Trap treatment from d 5 to 10
`showing the patchy (±) staining pat-
`tern in which some granulosa cells (black
`arrows) show nuclear staining, but oth-
`ers (red arrows) do not. I, Serial negative
`control section of H. J, Caspase-3 immu-
`nostaining of preantral follicles demon-
`strating one atretic (block arrow) and
`four healthy follicles (red arrows). K,
`Caspase-3 immunostaining of antral fol-
`licle confirming atresia with no staining
`of neighboring antral follicle (arrow). L,
`Capsase-3 staining of healthy antral fol-
`licle showing a single atretic cell (arrow).
`0, Oocyte; 0, granulosa cell; T, theca cell;
`A, antral cavity. Scale bar, 150 jm (B, F,
`and J-L) and 100 ban (A-D and G-U.
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`cells were very infrequent (Fig. 4D). There was no specific
`immunostaining in the negative control sections (Fig. 4E).
`
`The effect of VEGF inhibition on luteal HIF-la localization
`
`Nuclear HIF-la immunostaining was only rarely detected in
`the steroidogenic cells of fully functional mid- to late luteal
`corpora lutea (Fig. 5A). In vivo exposure to VEGF Trap in the
`midluteal phase was associated with the appearance of
`dear nuclear 1-HF-1a staining in some steroidogenic cells
`(Fig. 5, B-D). The proportion of steroidogenic cells with nuclear
`HIF-la expression increased with the duration of exposure to
`VEGF Trap (P C 0.05) (Fig. 6). There was never any specific
`staining in negative control sections (Fig. 5E). in addition to
`nuclear staining in steroidogenic cells after Trap treatment,
`
`nuclear HIF-1 a expression was evident in some luteal endo-
`thelial cells (Fig. 517). Endothelial cells never showed nuclear
`HIF-la localization in the corpus luteun-t in the absence of VEGF
`Trap treatment. Exposure to chorionic gonadotropin during
`early pregnancy did not result in an increased proportion of
`luteal cells with nuclear HIP-la staining than that seen in the
`functional corpus luteum of control cycles (Fig. 6).
`
`Discussion
`
`HIF-la is a transcription factor involved in the hypoxic
`regulation of VEGF expression (26). Its action is associated
`with nuclear localization of the protein, and as such, nuclear
`irnmunolocalization of the protein has been shown to be
`predictive of hypoxic signaling in tissues in which angio-
`
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`Duncan et at. • HIP-la in Primate Ovary
`
`Endocrinology, July 2008, 149(7):3313-3320 3317
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`
`the natural ovarian cycle supports a role for HIF-la in the
`regulation of physiological ovarian angiogenesis.
`During the follicular phase, nuclear HIP-la iniinunostain-
`ing was largely absent from the steroidogenic cells of the
`follicle. Indeed nuclear HIF-la staining was notably absent
`from the avascular granulosa cells of the dominant preovu-
`latory follicle, although notably at this stage, there is clear
`VEGF expression in the granulosa cells (7). During ovulation
`there was marked HIF-la staining in the luteinizing gran-
`ulosa cells. This immunostaining persisted into the early
`corpus luteum, a time during which there is intense angio-
`genesis that is regulated by VEGF (2). Indeed, we have pre-
`viously shown that VEGF niRNA is also highly expressed at
`this time and reduced when LH is removed using GnRH
`antagonist treatment (7). These observations are consistent
`with periovulatory hypoxia and a possible role for the
`HIF-la transcription factor in the regulation of the intense
`angiogenesis associated with luteal formation. There is little
`previous work on HIF-la localization in the ovary. In the pig
`ovary HIF-la mRNA was found in corpora lutes, and ex-
`pression tended to decrease as the corpus luteum matured
`(29). Again this is consistent with a role for HIP-la in luteal
`formation.
`The clear and coordinated changes in HIF-la localization
`during ovulation would, however, also be consistent with
`F-IIF-la localization being hormonally regulated. In addition
`to hypoxia (17), HIF-la expression can be ligand-stimulated
`under nonhypoxic conditions (30). It is therefore possible
`that HIF-la is directly stimulated by the LH surge as well as
`the local hypoxic environment. Whether this is a direct LH
`effect or secondary to the increased metabolic demands and
`glucose use induced by LH, and required for the marked
`change in steroidogenesis associated with luteinization, is
`not clear. What is clear is that there is a link between HIF
`expression and the energy requirements of the cell (31). That
`said, the large metabolic demands of the steroidogenic cells
`are maintained in the functioning corpus luteum, with its
`fully formed vascular network, in the absence of nuclear
`HIP-la staining. This suggests that there is a associated hyp-
`oxia during luteal formation but that this signal is facilitated,
`and regulated, by LH. Indeed, FSH has been shown to induce
`HIM in rat granulosa cells (32). In addition, there is evidence
`that HIF-2a expression in human luteinizing granulosa cells
`can be directly regulated by hCG in vitro under normoxic
`conditions (33).
`Outside the periovulatory period, however, the nuclear
`localization of FUF-la in the natural ovarian cycle is less
`marked. One time when VEGF is clearly required is in de-
`velopment of the follicular vasculature (2) and in the devel-
`oping primate follicle, the main source of VEGF during fol-
`licular growth is the avascular granulosa cells of the follicle
`(6). Although there was occasional nuclear staining in the
`follicles of the follicular phase, this was not particularly no-
`table despite clear VEGF expression (2). These cells, however,
`have the capacity to demonstrate nuclear HIP-I a localization
`when VEGF is withdrawn. Whether, in the presence of
`VEGF, HIP-la expression is important but transient such that
`nuclear localization is difficult to detect is not yet clear.
`Although hypoxia may have a role in follicular development,
`these studies, however, would suggest that HIF-la-indepen-
`
`A
`hfl 100
`0) C
`C
`a 80
`0) 0 C
`
`60
`
`40 1 213-
`
`B
`
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`05
`
`Contro
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`Trap
`00-10
`
`Trap
`05-10
`
`U + 0 ±
`
`C
`20-
`C
`Cu
`
`0
`Control Trap id Trap 2d Trap 4d
`
`LL
`
`FIG. 3. 3. HIP-la immunostaining in the granulosa cells of antral fol-
`licles (Cl mm). The nuclear HIP-la immunostaining pattern for each
`follicle was classified as intense in all granulosa cells (+), light or
`patchy in granulosa cells (±), or absent from granulosa cells (—). A,
`The immuriostaining pattern changes after VEGF Trap treatment in
`the follicular phase (P < 0.001, f) All healthy follicles in control
`ovaries collected in the midfollicular phase (d 5) (n = 77), the late
`follicular periovulatory phase (d 10) (a = 26), and from VEGF Trap-
`treated ovaries at 10 after 10 d (dO-iC) (n = 36) or it (d 5-10) (n =
`36) of treatment were scored. B, The immunostaining pattern changes
`after VEGF treatment in the midluteal phase (P C 0.001, f). All
`healthy follicles in luteal d 12 and 14 control ovaries (n = 90) and after
`VEGF Trap from luteal d 10 for 1 (id) (n = 45), 2 (2d) (n = 33), and
`4 (4d) (a = 20) days of treatment were scored.
`
`genesis is required such as prostatic cancer (27) and the
`endometrium (16). The ovary has marked, regulated, and
`cyclical angiogenesis (2) that is fundamental for follicle
`growth (6) and luteal formation and function (4, 28). This
`arigiogenesis is dependent on and regulated by gonadotro-
`pins (7) and VEGF (2). Here we investigated tissue hypoxia
`in the primate ovary by studying the nuclear localization of
`the HIP-la protein in the natural cycle and after VEGF in-
`hibition. The change in its nuclear immunolocalization across
`
`Mylan Exhibit 1112
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`Page 5
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`

`

`3318 Endocrinology, July 2003, 149(7):3313-3320
`
`Duncan et al. • HIF-la in Primate Ovary
`
`FIG, 4, Immunolocalization of HlF-lry
`in the marmoset corpus luteum. A, Lu-
`teinizing follicle in the periovulatory
`period showing intense nuclear immu-
`nostaining (arrows) in the granulosa
`(G) cells but not the theca (T) cells. B,
`Collapsing follicle forming the very
`early corpus luteum demonstrating
`continued nuclear staining in the lu-
`teinized granulosa cells (arrows). C, A
`midluteal corpus luteum showing faint
`cytoplasmic staining but no nuclear
`HIF-la immunostaining. D, Very late
`naturally regressing corpus luteum
`with cytoplasmic staining in steroido-
`genic cells and possible nuclear stain-
`ing in very few cells (arrows). B, Serial
`negative control section of B showing no
`staining. A, Collapsing antral cavity.
`Scale bar, 100 Am.
`
`dent regulation of VEGF maybe more important than during
`luteal formation. Indeed, treatment with CnRH antagonists
`during the follicular phase in the marmoset decreased the
`vasculature of the antral follicles but also decreased VEGF
`expression (7). This suggests a primary role for gonadotropin
`rather than hypoxic regulation of VEGF expression during
`folliculogenesis.
`Another time when there is up-regulation of VEGF ex-
`pression is during luteal rescue. In women maternal rec-
`ognition of pregnancy is associated with continued an-
`giogenesis (34) and increased secretion of VEGF under the
`
`influence of hCc (12). Here, however, early pregnancy
`was not associated with clear nuclear HIF-la staining in
`the corpus luteum. It is uncertain whether HIF-la really
`is not up-regulated in the fully vascularized corpus luteum
`by chorionic gonadotropin, or this represents a difference
`between marmosets and women. Although VEGF seems to
`be hormonally regulated in the marmoset corpus luteum,
`there is no increased angiogenesis during maternal rec-
`ognition of pregnancy (23), and, unlike women, increased
`VEGF may not be required during maternal recognition of
`pregnancy (23).
`
`C
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`
`Fie. 5. Immunolocalization of HIF-la
`in the marmoset corpus luteum after
`VEGF inhibition. A, Fully functional
`mid-luteal corpus luteum. B, Corpus lu-
`teum after 1 it exposure to VEGF Trap
`in vivo showing nuclear irnmunostain-
`ing (arrows). C, Increased luteal nu-
`clear HIP-1a staining (arrows) after 4 d
`of VEGF Trap treatment in vivo. D,
`Higher-power view of a corpus luteum
`of another animal, exposed to VEGF
`Trap for 4 d, showing nuclear immune-
`staining (arrows) in luteal steloido-
`genic cells. B, negative control section
`of D showing no staining. F, Another
`ovary after 4 d of VEGF trap treatment
`in the midluteal phase showing nuclear
`HIF-la staining in the endothelial cells
`of an associated small blood vessel (by).
`Scale bar, 100 Am (A-C) and 150 Am
`(P-F).
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`Mylan Exhibit 1112
`Mylan v. Regen