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`
`Endocrinology 143(7):2797–2807
`Copyright © 2002 by The Endocrine Society
`
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`Prevention of Thecal Angiogenesis, Antral Follicular
`Growth, and Ovulation in the Primate by Treatment
`with Vascular Endothelial Growth Factor Trap R1R2
`
`CHRISTINE WULFF, HELEN WILSON, STANLEY J. WIEGAND, JOHN S. RUDGE, AND
`HAMISH M. FRASER
`Medical Research Council (C.W., H.W., H.M.F.) Human Reproductive Sciences Unit, Edinburgh, United Kingdom EH3 9ET;
`Department of Obstetrics and Gynecology, University of Ulm (C.W.), 89075 Ulm, Germany; and Regeneron Pharmaceuticals,
`Inc. (S.J.W., J.S.R.), Tarrytown, New York 10591
`
`This study was designed to investigate the effects of inhibition
`of thecal angiogenesis on follicular development in the mar-
`moset monkey (Callithrix jacchus). To inhibit vascular endo-
`thelial growth factor (VEGF), a soluble combined truncated
`form of the fms-like tyrosine kinase (Flt) and kinase insert
`domain-containing receptor (KDR) receptor fused to IgG
`(VEGF Trap R1R2) was administered for 10 d during the fol-
`licular phase of the cycle. Changes in angiogenesis and fol-
`licular cell proliferation were quantified using immunocyto-
`chemistry for bromodeoxyuridine to obtain a proliferation
`index, CD31 to visualize endothelial cell area, and dual stain-
`ing to distinguish thecal endothelial cell proliferation. The
`effects of the treatment on follicular development were as-
`sessed by morphometric analyses by measuring follicle diam-
`eter, thecal thickness, and a proliferation index for granulosa
`cells. Follicular atresia was detected and quantified using the
`terminal deoxynucleotidyltransferase-UTP nick end labeling
`
`method. Effects on gene expression of VEGF and its receptors,
`Flt and KDR, were studied by in situ hybridization. VEGF
`Trap R1R2 treatment resulted in a significant decrease in
`thecal proliferation and endothelial cell area, demonstrating
`the suppression of thecal angiogenesis. The absence of a nor-
`mal thecal vasculature was associated with a significantly
`reduced thecal thickness. Antral follicular development was
`severely compromised, as indicated by decreased granulosa
`cell proliferation, decreased follicular diameter, and lack of
`development of ovulatory follicles. Furthermore, the rate of
`atresia was significantly increased. VEGF expression in gran-
`ulosa and thecal cells increased after treatment, whereas Flt
`and KDR expressions in thecal endothelial cells were mark-
`edly decreased. These results show that VEGF Trap treatment
`is associated with the suppression of follicular angiogenesis,
`which results in the inhibition of antral follicular develop-
`ment and ovulation. (Endocrinology 143: 2797–2807, 2002)
`
`THE OVARY IS distinctive in being a site of active an-
`
`giogenesis in the adult. Angiogenesis takes place in the
`developing follicle before ovulation and in the corpus luteum
`formed postovulation (1– 4). It is now well established that
`active cyclical angiogenesis plays a key role in normal luteal
`function (1, 4). However, its contribution to normal follicular
`growth and function has not been addressed experimentally.
`Thus, little is known about the direct relevance of the thecal
`vasculature for follicular growth, development, and atresia.
`Although small preantral follicles are avascular, angiogen-
`esis is initiated during early follicular development and con-
`tinues throughout follicular growth. The vascular sheath that
`develops during follicular maturation in the thecal compart-
`ment expands with ongoing folliculogenesis (3). The thecal
`capillaries do not penetrate the membrana propria, so the
`granulosa compartment remains avascular until breakdown
`of the basement membrane at ovulation.
`The vasculature of the follicle is thought to be necessary for
`the delivery of hormones, hormone precursors, oxygen, and
`nutrients. It has been suggested that the preferential delivery
`of gonadotropins via a more highly developed vascular sys-
`
`Abbreviations: BrdU, Bromodeoxyuridine; Flt, fms-like tyrosine ki-
`nase; KDR, kinase insert domain-containing receptor; NBT, nitro blue
`tetrazolium; TBS, Tris-buffered saline; TUNEL, terminal deoxynucle-
`otidyltransferase-UTP nick end labeling; VEGF, vascular endothelial
`growth factor.
`
`tem in individual follicles plays an instrumental role in the
`selection and growth of the dominant follicle (5). The rela-
`tionship between changes in angiogenesis and onset of atre-
`sia is uncertain due to difficulties in determining the tem-
`poral relationship between these processes, but decreased
`vascularity in atretic follicles has been reported in a number
`of species (5–7), including the marmoset (3).
`With regard to the molecular mechanisms controlling follic-
`ular angiogenesis, the presence of the vascular endothelial
`growth factor (VEGF), a principal angiogenic factor, has been
`described in the ovarian follicle (8, 9). More recently, we have
`demonstrated by direct inhibition of VEGF in vivo in the primate
`that VEGF is a major regulator of follicular angiogenesis (3).
`Inhibition of VEGF was followed by a severe restriction of thecal
`angiogenesis in the developing follicle. To investigate the im-
`portance of the thecal vasculature for follicular maturation, the
`approach of suppressing thecal angiogenesis by in vivo inhibi-
`tion of VEGF was used in the current study. A new compound,
`VEGF Trap R1R2, comprising the extracellular domain of the
`two VEGF receptors, VEGF-R1 (Flt) (fms-like tyrosine kinase)
`and VEGF-R2 (KDR) (kinase insert domain-containing recep-
`tor), was administered to marmoset monkeys throughout the
`follicular phase. The efficacy of VEGF Trap R1R2 to suppress
`thecal angiogenesis was tested using bromodeoxyuridine
`(BrdU) immunocytochemistry as a proliferation marker, CD31
`as a specific endothelial cell marker and dual staining to dis-
`
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`2798 Endocrinology, July 2002, 143(7):2797–2807
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`FIG. 1. Structure of VEGF receptors and the VEGF Trap. VEGF-R1
`(A) and VEGF-R2 (B) both contain seven extracellular domains, which
`differ between the two receptors. These extracellular domains are
`responsible for VEGF binding. The soluble VEGF Trap R1R2 (C) was
`created by fusion of domain 2 of VEGF-R1 and domain 3 of VEGF-R2
`with the FC portion of IgG.
`
`jected to immunocytochemistry, in situ hybridization, and TUNEL. Tis-
`sue sections were dewaxed in xylene, rehydrated in descending con-
`centrations of ethanol, washed in distilled water, and stained with
`hematoxylin (Richard-Allan, Richland, MI) for 5 min, followed by a
`wash in water and acetic alcohol before staining with eosin (Richard-
`Allan) for 20 sec. After dehydrating in ascending concentrations of
`ethanol and xylene, sections were mounted.
`
`Immunocytochemistry
`
`The effects of the treatment on the establishment of the thecal vascular
`network were studied by 1) quantifying the number of proliferating cells
`stained for BrdU, 2) identifying endothelial cells using CD31 staining,
`and 3) distinguishing proliferating endothelial from nonendothelial cells
`by colocalization of BrdU and CD31.
`For BrdU and CD31 immunostaining, antigen retrieval was per-
`formed by pressure cooking (Tefal Clypso pressure cooker, Tefal, Essex,
`UK) sections in 0.01 m citrate buffer, pH 6, for 6 min at high pressure
`setting 2. Slides were then left for 20 min in hot buffer and washed in
`TBS (0.05 mol/liter Tris and 9 g/liter NaCl). To reduce nonspecific
`binding sections were blocked in normal rabbit serum (1:5 diluted in TBS
`containing 5% BSA) for 30 min. Primary antibodies CD31 (mouse an-
`tihuman CD31, DAKO Corp., Copenhagen, Denmark) or BrdU (mouse
`anti-BrdU, Roche Molecular Biochemicals) were diluted 1:20 or 1:30 in
`TBS, respectively. Incubation was carried out overnight at 4 C. Slides
`were washed three times in TBS. Incubation with the secondary anti-
`body, rabbit antimouse Ig (1:60 diluted in NRS:TBS; DAKO Corp.), was
`performed for 40 min at room temperature, followed after two washes
`in TBS by incubation of the alcaline phosphatase-antialcaline phospho-
`lase (APAAP) complex (1:100 dilution in TBS, DAKO Corp.) for 40 min
`at room temperature. Visualization was performed using 500 ␮l/slide
`nitro blue tetrazolium (NBT) solution containing 45 ␮l NBT substrate
`(Roche Molecular Biochemicals), 10 ml NBT buffer, 35 ␮l 5-bromo-3-
`chloro-3-indolyl-phosphate, and 10 ␮l levamisole. Sections for BrdU
`
`tinguish between proliferating endothelial and nonendothelial
`cells. The influence on follicular development was assessed by
`morphological and morphometric image analyses as well as
`quantification of granulosa cell proliferation. As granulosa
`cells of the follicle die of apoptosis during the process of
`atresia (10), the terminal deoxynucleotidyltransferase-UTP
`nick end labeling (TUNEL) method was used to detect apo-
`ptotic granulosa cells for definite classification and quantifica-
`tion of atretic follicles. The effects of inhibition of VEGF on the
`expression of VEGF and its receptors Flt and KDR was inves-
`tigated using in situ hybridization.
`
`Materials and Methods
`VEGF Trap R1R2
`
`The VEGF Trap R1R2 used in these experiments is a recombinant
`chimeric protein comprising portions of the extracellular, ligand binding
`domains of the human VEGF receptors Flt-1 (VEGF-R1, Ig domain 2) and
`KDR (VEGF-R2, Ig domain 3) expressed in sequence with the Fc portion
`of human IgG (Fig. 1). The presence of the Fc domain results in ho-
`modimerization of the recombinant protein, thereby creating a high
`affinity (KD1–5pM) VEGF Trap.1 The VEGF trap was expressed in CHO
`cells and was purified by protein A affinity chromatography followed
`by size-exclusion chromatography. The specificity of VEGF binding and
`the affinity to VEGF of VEGF Trap R1R2 were determined by Biacore
`(Uppsala, Sweden).
`
`Animals
`
`Adult female common marmoset monkeys (Callithrix 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 (11). Blood samples
`were collected three times per week by femoral venipuncture without
`anesthesia, and plasma was subjected to progesterone assay as described
`previously (4).
`
`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. To synchronize timing of ovulation during
`the pretreatment cycle, marmosets were given PGF2␣ in the mid to late
`luteal phase to induce luteolysis. In the late luteal phase of the subse-
`quent cycle, four marmosets were treated with VEGF trap at a dose of
`25 mg/kg, injected sc on d 0, 2, 4, 6, and 8 of the follicular phase. Ovaries
`were collected 2 d later on d 10 of the cycle. Eleven control marmosets
`were studied (four on d 1–2, three on d 7– 8, and four at d 11 of the cycle).
`Control animals were treated with vehicle containing 5 mm phosphate,
`5 mm citrate, 100 mm sodium chloride, 0.1% (wt/vol) Tween 20, and 20%
`(wt/vol) sucrose. All animals were injected iv with 20 mg BrdU (Roche
`Molecular Biochemicals, Essex, UK) in saline 1 h before being sedated
`using 100 ␮l ketamine hydrochloride (Parke-Davis Veterinary, Ponty-
`pool, UK) and killed with an iv injection of 400 ␮l Euthetal (sodium
`pentobarbitone, Rhone Merieux, Harlow, UK). After cardiac exsangui-
`nation via a heparinized syringe, ovaries were removed immediately
`and fixed in 4% neutral buffered formalin. After 24 h, the ovaries were
`put into 70% ethanol, dehydrated, and embedded in paraffin according
`to standard procedures.
`
`Hematoxylin-eosin staining
`
`The embedded ovaries were serially sectioned, and tissue sections (5
`␮m) were placed onto BDH SuperFrost slides (BDH, Merck & Co., Inc.,
`Poole, UK). For morphological and morphometric analyses every 20 of
`a total of 200 sections/ovary were used. Sections in between were sub-
`
`1 The detailed molecular structure and how it was created are de-
`scribed in the patent REG 710-A-PCT, VEGF Trap Application published
`December 2000, Publication WO 00/75319 A1.
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`Endocrinology, July 2002, 143(7):2797–2807 2799
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`were counterstained with hematoxylin, whereas sections for CD31 were
`not counterstained, so that quantitative image analysis could be per-
`formed. For dual labeling, slides were incubated first with CD31 and
`visualization with Fast Red [Sigma, Poole, UK; 1 mg Fast Red in 1 ml Fast
`Red buffer (20 mg naphtol AS-MX phosphate, 2 ml dimethyl formamide,
`and 98 ml 0.1 m Tris, pH 8.2)]. After staining for CD31, incubation with
`BrdU was performed. BrdU-stained cells were visualized with NBT as
`described above.
`
`In situ hybridization
`
`In situ hybridization was performed as described previously (4, 12).
`As the marmoset shows 97–98% homology of the known gene sequence
`with human genome, cRNA probes for human VEGF, Flt, and KDR were
`used. Sense and antisense probes were prepared using an RNA tran-
`scription kit (Ambion, Inc. Austin, TX) and were labeled with [35S]-
`uridine 5[prime]-triphosphate (NEN Life Science Products, Boston,
`MA). Deparaffinized sections were treated with 0.1 n HCl and then
`digested in proteinase K (5 ␮g/ml; Sigma) for 30 min at 37 C. After
`prehybridization for 2 h at 55 Csubsequent hybridization was per-
`formed in a moist chamber overnight. High stringency posthybridiza-
`tion washings and ribonuclease treatment were used to remove excess
`probe. Slides were then dehydrated, dried, and dipped in Ilford G5
`liquid emulsion (H. A. West, Edinburgh, UK). Exposure times for VEGF,
`Flt, and KDR were 4, 7, and 7 wk, respectively. Slides were subsequently
`developed (D19 developer, Kodak, Rochester, NY) and fixed (GBS,
`Kodak). All slides were counterstained with hematoxylin (Richard-
`Allan, Richland, MI), dehydrated, and mounted.
`
`In situ TUNEL
`
`It is known that during atresia granulosa cells undergo programmed
`cell death, apoptosis. Hence, the TUNEL method for detection of apo-
`ptotic cells was used to identify atretic follicles. Dewaxed and rehy-
`drated slides were incubated for 6 min in 20 ␮g/ml proteinase K (Sigma)
`at room temperature and blocked with normal sheep serum (1:5 dilu-
`tion). Slides were washed three times in TBS. For 3⬘-end labeling the TdT
`Kit (Roche Molecular Biochemicals) was used. 3⬘-OH ends of DNA
`fragments were labeled with 1 nm digoxigenin-11-deoxy-UTP (Roche
`Molecular Biochemicals) for 1.5 h at 37 C by 1 IU/␮l TdT (Roche Mo-
`lecular Biochemicals) in 50 ␮l buffer [30 mm Tris-HCl (pH 7.2), 140 mm
`sodium cacodylate, and 1.5 mm CoCl2; Roche Molecular Biochemicals).
`Negative control slides had the TdT replaced by the equivalent amount
`of buffer. Three rinses of the slides with TBS were followed by incubation
`with alkaline phosphatase-conjugated sheep anti-digoxigenin antibod-
`ies (1:100 dilution; Roche Molecular Biochemicals) in TBS for 90 min at
`room temperature. The labeling was visualized with NBT as described
`above. After air-drying, slides were put into xylene and mounted.
`
`Analysis of data
`
`Quantitative analysis was performed using an image analysis system
`linked to an Olympus Corp. camera, and the data were processed using
`Image-Pro Plus version 3.0 for Windows (Microsoft Corp.).
`
`Morphological characterization of ovarian follicles
`
`Stages of follicular development were defined as follows: primary
`follicles (containing only one granulosa cell layer), early secondary fol-
`licles (two to four granulosa cell layers, no antrum), late secondary
`follicles (more than four granulosa cell layers, no antrum), tertiary fol-
`licles (follicles containing an antrum), and ovulatory follicles (large
`antral follicles, ⬎2 mm). Follicles were classified as healthy if they
`contained a normal-shaped oocyte surrounded by granulosa cells that
`were regularly apposed on an intact basement membrane with normal
`appearance of granulosa cell nuclei without signs of pycnosis. Follicles
`not fulfilling these criteria were classified as unsuitable for analyses.
`Only follicles with a visible oocyte containing a nucleus were considered
`to ensure proper follicular classification.
`
`Morphometric analyses
`
`Serial sections of both ovaries of each animal stained for hematoxylin-
`eosin were subjected to morphometric analyses (10 sections at a distance
`
`of 100 ␮m/ovary from each other). A total of 31 primary, 523 secondary,
`and 181 tertiary follicles were analyzed in controls, and 29 primary, 327
`secondary, and 77 tertiary follicles were analyzed in treated animals. The
`image analysis system was set up to measure two diameters of the
`follicles in a right angle. From these diameters the mean follicular di-
`ameter was calculated. Furthermore, the thecal compartment was out-
`lined, and the mean thecal thickness was measured.
`
`Quantification of immunocytochemistry, in situ
`hybridization, and 3⬘-end labeling
`
`From our previous study (3) it was known that angiogenesis is ini-
`tiated in follicles containing more than four granulosa cell layers. Thus,
`in this study only follicles with four or more granulosa cell layers were
`analyzed. In all follicles the whole cross-sections were analyzed. Cap-
`tured images were thresholded, and the thecal and granulosa cell com-
`partments were outlined and analyzed separately.
`
`BrdU labeling
`Four sections per ovary were analyzed under ⫻200 magnification.
`The image analysis system was set up to measure the number of dark-
`stained nuclei (BrdU positive), and the number of dark- and light-
`stained nuclei (total number of cells) in the outlined compartment of
`interest. A proliferation index (i.e. BrdU-positive cells expressed as a
`percentage of the total number of cells) was calculated in the thecal and
`granulosa compartments for each follicle. The proliferation index was
`expressed as a mean value for the number of follicles assessed within
`each follicular stage and per animal.
`The automated image analysis of BrdU in the granulosa of secondary
`follicles failed to reliably distinguish between single cells because gran-
`ulosa cells have only a small cytoplasmic volume, so that the nuclei of
`different cells are in close vicinity. Thus, the granulosa cell proliferation
`index in these follicles was obtained by manual counting using an
`eyepiece with a grid.
`
`CD31 labeling
`
`The endothelial cell area (i.e. CD31-positive cells) was measured at
`⫻200 magnification in four sections of each ovary. The captured gray
`scale image was thresholded and converted to a binary image. The whole
`area of the thecal compartment and the CD31-positive area within the
`compartment was measured. The CD31-positive area was then calcu-
`lated per unit area of the thecal compartment and expressed as a mean
`value for the number of follicles assessed within each follicular stage and
`per animal.
`
`3⬘-End labeling
`
`Four sections per animal were analyzed. Follicles were classified as
`atretic if more than approximately 20% of the granulosa cells were
`apoptotic. The number of atretic and healthy late secondary and antral
`follicles were counted manually, and an index for atresia (atretic follicles
`expressed as a percentage of total number of follicles) was calculated.
`
`Statistical analysis
`
`Data obtained for different cycle and follicular stages were tested for
`significant differences using ANOVA, followed by Duncan’s multiple
`range test. Effects of the treatment compared with late follicular controls
`were determined using a two-tailed, unpaired t test. Differences were
`considered to be significant at P ⬍ 0.05. The tests were performed using
`SPSS version 6.1 for Macintosh (SPSS, Inc., Chicago, IL). All values are
`given as the mean ⫾ sem.
`
`Results
`Efficacy of the treatment to suppress thecal angiogenesis
`Dual staining for BrdU and CD31 (Fig. 2A) showed the
`established microvasulature in controls. Besides numerous
`single-stained cells for BrdU, some BrdU-positive cells were
`also associated with CD31 staining, indicating proliferating
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`2800 Endocrinology, July 2002, 143(7):2797–2807
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`Wulff et al. (cid:127) Inhibition of Follicular Development
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`FIG. 2. Dual staining for CD31 (red
`staining) and BrdU (black nuclei) in a
`follicle of a control (A) and an R1R2-
`treated animal (B). Note the reduction
`of proliferating cells (single and dual
`stained) and endothelial staining in the
`theca (Th) after treatment. Quantifica-
`tion of thecal proliferation (C) revealed
`a significant decrease in late secondary
`(LS) and tertiary (T) follicles. In these
`follicle stages the endothelial cell area
`(D) was also significantly reduced, dem-
`onstrating the suppression of thecal an-
`giogenesis after treatment. Different
`letters indicate significant differences.
`f, controls; u, R1R2 treated. ES, Early
`secondary follicle. Bar, 100 ␮m.
`
`endothelial cells. After R1R2 treatment (Fig. 2B), very few
`proliferating cells were visible, and thecal CD31 staining was
`markedly reduced. These observations were confirmed by
`quantitative analyses (Fig. 2, C and D). The thecal prolifer-
`ation index in late secondary follicles was 13.6 ⫾ 2.8% in early
`follicular controls, 13.2 ⫾ 1.8% in late follicular controls, and
`13.4 ⫾ 1.4% in early luteal controls. After treatment, a 79%
`reduction (P ⬍ 0.05) of thecal proliferation was found in late
`secondary follicles (3.4 ⫾ 0.6%). The thecal proliferation in-
`dex of tertiary follicles was 10.6 ⫾ 0.4% in early follicular
`controls, 15.1 ⫾ 2.1% in late follicular controls, and 16.2 ⫾
`2.0% in early luteal controls. After R1R2 treatment, a major
`92% reduction (P ⬍ 0.001) in thecal proliferation in tertiary
`follicles (1.1 ⫾ 0.2%) was observed.
`The endothelial cell areas measured in secondary or ter-
`tiary follicles of control animals were comparable (in sec-
`ondary follicles, 0.09 ⫾ 0.01 ␮m2/unit area during the early
`follicular phase, 0.09 ⫾ 0.008 ␮m2/unit area during the late
`follicular phase, and 0.09 ⫾ 0.008 ␮m2/unit area during the
`early luteal phase; in tertiary follicles, 0.2 ⫾ 0.01 ␮m2/unit
`area during the early follicular phase, 0.2 ⫾ 0.03 ␮m2/unit
`area during the late follicular phase, and 0.18 ⫾ 0.02 ␮m2/
`unit area during the early luteal phase). After R1R2 treatment
`a significant reduction of the endothelial cell area of 72% was
`found in secondary follicles (0.025 ⫾ 0.005 ␮m2/unit area)
`and 80% in tertiary follicles (0.04 ⫾ 0.007 ␮m2/unit area). In
`summary, it was evident that the treatment efficiently sup-
`pressed thecal angiogenesis in secondary and tertiary
`follicles.
`
`Effects of R1R2 treatment on thecal development
`Thecal development is initiated during early follicular
`growth when a follicle contains more than two granulosa cell
`layers. The fully differentiated theca is shown in Fig. 3A.
`Thecal cells have an elongated flat appearance surrounding
`the granulosa compartment in several layers. Fibrocytes are
`dispersed within these layers as the microvasculature is es-
`tablished. After R1R2 treatment (Fig. 3B), thecal cells ap-
`peared swollen, contained enlarged nuclei, and had lost their
`elongate shape. Furthermore, the theca lacks a microvascu-
`lature. In contrast, the occurrence of fibrocytes appeared
`unaffected.
`By measuring thecal thickness and plotting against the
`follicular diameter (Fig. 3C), a significant linear correlation
`was found for secondary follicles in controls (r ⫽ 0.9; P ⬍
`0.001) and treated animals (r ⫽ 0.86; P ⬍ 0.001). However,
`after R1R2 treatment the slope of the curve was reduced,
`indicating that at a given follicle diameter the theca is thinner
`than that in controls. Comparison of the mean thecal thick-
`ness in these follicles confirmed a significant reduction (P ⬍
`0.05) in thecal thickness after treatment. No correlation be-
`tween thecal thickness and follicle diameter was observed in
`tertiary follicles.
`
`Effects of R1R2 treatment on the expression of VEGF and
`its receptors KDR and Flt
`In controls, VEGF mRNA was expressed in the granu-
`losa and to a lesser extent in the thecal compartment in
`secondary and tertiary follicles (Fig. 4A). An increase in
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`FIG. 3. Effects of the inhibition of thecal angiogenesis on thecal development. A, A hematoxylin- and eosin-stained section of the theca (Th)
`of a control. Thecal cells have an elongated, flattened shape. Capillaries pass through the thecal compartment. B, Thecal cells after R1R2
`treatment appear swollen with enlarged nuclei. Note the lack of the thecal capillaries (bar, 100 ␮m). Measuring the thecal thickness and plotting
`it against the follicle diameter (C), a significant linear correlation for both in controls and after R1R2 treatment was found. However, the curve
`for the R1R2 treatment exhibited a lower slope, indicating that the theca after treatment is thinner. This was confirmed by comparison of the
`mean thecal diameters (D). Different letters indicate significant differences. LF, Late follicular control.
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`FIG. 4. Effects of the treatment on the gene expression of VEGF (A and B), KDR (C and D), and Flt (E and F). After treatment, VEGF was
`expressed in increasing amounts in the thecal and granulosa cell compartments of secondary and tertiary follicles. KDR (B) and Flt (E) mRNA
`was localized in thecal endothelial cells, and after treatment a complete down-regulation was notable (D and F). Bar, 50 ␮m.
`
`VEGF expression in the granulosa was observed in the
`preovulatory follicles. After R1R2 treatment (Fig. 4B)
`VEGF mRNA expression was markedly increased in the
`granulosa of secondary and tertiary follicles. Expression in
`the theca was also increased, especially in secondary fol-
`licles, such that the expression in the theca appeared to be
`higher than that in the granulosa. The KDR receptor
`mRNA (Fig. 4C) and Flt receptor mRNA (Fig. 4E) are both
`expressed in the endothelium of the thecal vasculature of
`both secondary and tertiary follicles. A complete down-
`
`regulation of both receptors (Fig. 4, D and F) was found
`after R1R2 treatment. This decrease was attributable not
`only to the dearth of thecal endothelial cells after treat-
`ment, but also to a reduction in expression in the remain-
`ing cells.
`
`Secondary effects of the treatment on follicular development
`
`By gross inspection, it appeared that after R1R2 treatment
`ovaries were smaller than controls. No signs of an ovulatory
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`Endocrinology, July 2002, 143(7):2797–2807 2803
`
`follicle or an ovulation stigma were found, which were ob-
`served in late follicular or early luteal controls. A significant
`decrease in ovarian weight (37 ⫾ 6.9 mg) after treatment was
`measured compared with early follicular (113 ⫾ 12 mg), late
`follicular (70 ⫾ 13 mg), and early luteal (127 ⫾ 35 mg) controls.
`In a cross-section through a typical control ovary of the
`early follicular phase (d 1–2; Fig. 5A) the regressing luteal
`tissue of the previous cycle was visible, occupying the ma-
`jority of the organ. Numerous healthy and atretic small and
`medium sized antral follicles were also present. In the late
`follicular phase ovary (d 7– 8; Fig. 5B) the predominant fea-
`ture was the developed preovulatory follicles (two or three
`per animal). Besides these, a number of smaller antral folli-
`cles are located within the cortex. After ovulation (d 10 –11;
`Fig. 5C), the early corpus luteum dominates, occupying two
`thirds of the ovary. Medium and large antral follicles, a
`number of them either luteinized or atretic, are present
`within the cortex. After R1R2 treatment, the most striking
`observation is the absence of medium and large antral fol-
`licles (Fig. 5D). A small proportion of regressed luteal tissue
`from the previous cycle was visible, but fresh, healthy cor-
`pora lutea were absent.
`Depending on the stage of follicular development, gran-
`ulosa cell proliferation was also effected by the treatment. In
`secondary follicles of controls (Fig. 6A) numerous granulosa
`cells were BrdU positive, indicating active cell proliferation.
`After R1R2 treatment no differences were apparent (Fig. 6B).
`In tertiary follicles of controls (Fig. 6C) a large number of
`
`granulosa cells are also proliferating, whereas after treatment
`(Fig. 6D) the number of proliferating granulosa cells mark-
`edly decreased. Quantification (Fig. 6E) confirmed these ob-
`servations, showing a nonsignificant decrease in secondary
`follicles and a significant reduction in the proliferation index
`after R1R2 treatment in tertiary follicles.
`The results of granulosa cell proliferation are consistent
`with morphometric analysis of follicular diameter. There
`was no difference in follicle diameter of early follicular
`stages (primary and early secondary follicles). Also no
`differences were observed for late secondary follicles be-
`tween early follicular phase (167 ⫾ 9.1 ␮m), late follicular
`phase (174 ⫾ 8.1 ␮m), early luteal phase (161 ⫾ 5.2 ␮m),
`and after R1R2 treatment (171 ⫾ 5 ␮m). However, major
`differences were detectable in tertiary follicles. Frequency
`measurements revealed that in controls, the peak fre-
`quency of tertiary follicles was 950 ␮m (Fig. 7A). A smaller
`number of large antral follicles (⬎1300 ␮m) and ovulatory
`follicles (⬃2500 ␮m) were also present. In comparison, in
`R1R2-treated animals (Fig. 7B) the peak frequency was
`reduced (750 ␮m). Most striking was the absence of fol-
`licles over 1000 ␮m. Comparison of the mean follicular
`diameter of tertiary follicles (Fig. 7C) showed a signifi-
`cantly decreased diameter in follicles after R1R2 treatment
`(722 ⫾ 13 ␮m) compared with controls (973 ⫾ 31 ␮m).
`The TUNEL method to detect apoptosis in follicles to clas-
`sify them definitely as atretic (i.e. the percentage of apoptotic
`follicles of the total number of follicles) showed that atresia
`
`FIG. 5. Effects of the inhibition of thecal angiogenesis on follicular development. Hematoxylin- and eosin-stained sections of the early follicular
`phase (A), late follicular phase (B), and early luteal phase (C) and after R1R2 treatment (D) are shown. Note the lack of large antral follicles
`after treatment compared with all other stages. Bar, 200 ␮m.
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`Wulff et al. (cid:127) Inhibition of Follicular Development
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`FIG. 6. Effects of the treatment on granulosa cell
`proliferation as indicated by BrdU immunocyto-
`chemistry. A, A late secondary follicle (LS) of a
`control ovary; B, a comparable follicle after treat-
`ment. No obvious differences in BrdU staining
`(black nuclei) are visible. C and D, A tertiary
`follicle of a control and R1R2-treated ovary is
`shown, respectively. Note the decline in granu-
`losa cell proliferation after the treatment (bar,
`100 ␮m). Quantitative analyses (E) confirmed the
`observation of decreased granulosa cell prolifer-
`ation in antral follicles after treatment (u) com-
`pared with controls (f).
`
`was rarely detectable in follicles up to the late secondary
`stage, and no quantitative difference in atresia was found
`between controls and R1R2-treated animals. In tertiary fol-
`licles, atresia did not vary between the normal cycle phases,
`being 37 ⫾ 1.6% during the early follicular phase, 35 ⫾ 6.8%
`during the late follicular phase, and 41 ⫾ 4.3% during the
`early luteal phase. However, after R1R2 treatment, a signif-
`icant doubling (78 ⫾ 4%) in the atretic rate was found com-
`pared with all controls (Fig. 7D).
`
`Discussion
`This study has established the importance of the thecal
`vasculature for follicular develop