Graphical Abstract

   graphic file with name fx1.jpg
   [41]Open in a new tab

Highlights

     * •
       p73 is required for murine ovarian folliculogenesis and proper
       corpus luteum formation
     * •
       p73 loss leads to defects in progesterone signaling and mammary
       gland branching
     * •
       In murine ovaries, p73 is expressed specifically in granulosa cells
     * •
       p73 regulates components of the granulosa cell focimatrix and
       migration
     __________________________________________________________________

   Molecular Network; Functional Aspects of Cell Biology; Developmental
   Biology; Omics

Introduction

   The p53 family of proteins, p53, p63, and p73, are sequence-specific
   transcription factors that are required for cell cycle control, DNA
   repair, apoptosis, adhesion, organ development, and cell
   differentiation ([42]Kaghad et al., 1997, [43]Schmale and Bamberger,
   1997, [44]Yang et al., 1998, [45]Osada et al., 1998, [46]Trink et al.,
   1998, [47]Holembowski et al., 2014). All three p53 family members share
   a high degree of structural and amino acid sequence similarities within
   their transactivation domains, DNA binding domains, and oligomerization
   domains ([48]Harms and Chen, 2006, [49]Dotsch et al., 2010), which
   accounts for similar genomic binding sites and regulation of
   overlapping target genes. Unlike p53, p63 and p73 are transcribed from
   two separate promoters that encode functionally divergent variants. The
   transcriptionally active (TA) isoform encodes the full-length protein,
   whereas the alternative transcript (ΔN) encodes an isoform lacking the
   amino-terminal transactivation domain ([50]Kaghad et al., 1997,
   [51]Yang et al., 1998). Thus, ΔNp63 and ΔNp73 isoforms act as
   dominant-negative regulators of TAp63 and TAp73 ([52]Yang et al., 1998,
   [53]Stiewe et al., 2002, [54]Grob et al., 2001).

   p63 and p73 play important roles in cell differentiation and tissue
   development. p63 is a key regulator of ectodermal differentiation and
   stratification of the epidermis. Mice lacking p63 fail to develop
   stratified epithelia, exhibit defective limb and glandular epithelial
   development, and die shortly after birth due to dehydration ([55]Mills
   et al., 1999, [56]Yang et al., 1999). Mice deficient for all isoforms
   of p73 exhibit runting, sterility, hippocampal dysgenesis and
   hydrocephalus, as well as chronic infection and inflammation in the
   lungs, sinus, and ears ([57]Yang et al., 2000). The development of
   p73-isoform-specific knockout mouse models provided significant insight
   into the roles of select p73 isoforms. TAp73-deficient mice exhibit
   sterility, hippocampal dysgenesis, hydrocephalus, premature aging,
   genomic instability, and increased frequency of tumors ([58]Tomasini
   et al., 2008). In contrast, mice that lack ΔNp73 are fertile and
   display signs of neurodegeneration, including hippocampal dysgenesis
   and hydrocephalus ([59]Wilhelm et al., 2010). Thus the sterility
   defects observed in the global p73-deficient animals are due to a
   deficiency in the TAp73 isoform. Recently, our laboratory ([60]Marshall
   et al., 2016) and others ([61]Nemajerova et al., 2016) discovered that
   TAp73 is required for multiciliated cell differentiation and acts as a
   transcriptional regulator of a gene network required for ciliogenesis.
   The discovery provided mechanistic insight into the diverse phenotypes
   observed in p73-deficient mouse models. Impaired cilia formation in
   p73-deficient mice leads to insufficient clearance of pathogens from
   the lungs and sinuses causing chronic inflammation. Furthermore, loss
   of cilia in reproductive tissues decreases transport of the sperm and
   ova through epididymis and fallopian tubes, respectively, leading to
   infertility.

   Other phenotypes of p73-deficient mice have been reported that are
   likely to originate from processes unrelated to ciliogenesis. Male mice
   that lack TAp73 exhibit increased DNA damage and apoptosis in
   spermatogonial cells within the testes, which results in defective germ
   cell maturation and differentiation, required for proper
   spermatogenesis ([62]Inoue et al., 2014, [63]Holembowski et al., 2014).
   TAp73-deficient female mice exhibit meiotic spindle formation
   abnormalities during oocyte maturation and impaired ovulation
   ([64]Tomasini et al., 2008). We report herein that p73 expression in
   the ovarian follicle, the structure in which the oocyte develops, is
   critical for oocyte development, ovulation, and fertility.
   Specifically, p73 is required in granulosa cells for the expression of
   a p73-dependent gene set that regulates cell adhesion and migration,
   including genes that encode key components of granulosa-cell-associated
   focimatrix.

Results

p73 Is Required for Murine Ovarian Follicle Maturation

   We analyzed ovaries from age-matched nulliparous female mice
   genetically engineered to lack functional p73 in all tissues (referred
   to as p73−/− hereafter and described in [[65]Marshall et al., 2016]).
   Histological analysis of ovaries in 12-week-old p73+/+ female mice
   confirmed the presence of corpus luteum, the final stage of the mature
   ovarian follicle, suggesting that follicle maturation and ovulation
   were ongoing in mice at this age. p73+/+ ovaries harbored an average of
   12 luteal structures per ovary, whereas age-matched p73−/− ovaries had
   an average of 1 luteal structure per ovary (p value <0.01) ([66]Figures
   1A and 1B). We quantified the total number of follicles per ovary at
   various stages of development (primordial, primary, secondary, and
   antral stages) using methodology as previously described ([67]Kim
   et al., 2015) ([68]Figures S1A and S1B). We observed a 35% decrease in
   the number of primary follicles (p value <0.001), a 59% decrease in the
   number of secondary follicles (p value <0.001), and a 49% decrease in
   the number of antral follicles (p value <0.001), but no significant
   difference in the number of primordial follicles in p73−/− mice
   compared with p73+/+ littermates ([69]Figure 1C).

Figure 1.

   [70]Figure 1
   [71]Open in a new tab

   p73 Is Required for Murine Ovarian Follicle Development

   (A) Representative H&E images of p73+/+ and p73−/− ovaries (scale bars,
   400 μm and 100 μm, respectively). Arrowheads and labels represent
   corpora lutea (CL) and different stages of follicle development: Prd,
   primordial; Prm, primary; Sec, secondary; Ant, antral follicles.

   (B and C) Each data point represents the average of two independent
   manual quantifications of six ovaries per genotype. Bars represent the
   mean. (B) Total number of corpus luteum per ovary (C) Total number of
   follicles per ovary. **p value< 0.01, ***p value < 0.001.

   (D and E) Representative H&E (asterisks represent granulosa cells and
   arrowheads represent theca cells) and IF images of p73+/+ and p73−/−
   ovaries show that (D) p73 (red) co-localizes with granulosa cell marker
   FOXL2 (green) in the follicles and (E) p73 (red) is not expressed in
   theca cells, which are stained by CYP17A1 (green) (scale bar, 50 μm).

   See also [72]Figure S1.

   Analysis of p73 protein levels in murine ovaries, at 6, 9, 12, and
   16 weeks of age, confirmed that full-length p73 protein was expressed
   in ovaries collected at all time points in p73+/+ animals and was not
   expressed in p73−/− animals ([73]Figure S1C). To determine the
   localization of p73 protein within the ovary, we used well-established
   cell markers to stain follicular granulosa cells (FOXL2) ([74]Schmidt
   et al., 2004) and follicular theca cells (CYP17A1) ([75]Park et al.,
   2010). Through immunofluorescence (IF) staining, p73 was expressed in
   FOXL2-positive granulosa cells ([76]Figure 1D, top panel) but not in
   CYP17A1-positive theca cells ([77]Figure 1E, top panel). Although the
   number of follicles were reduced in the absence of p73, we found that,
   in cases in which follicles were able to form in p73−/− ovaries, the
   lack of p73 did not block the formation of the granulosa or theca cell
   layers in the follicle ([78]Figures 1D and 1E, lower panels). We
   further analyzed p73 expression in human ovaries using data obtained
   from the Genotype-Tissue Expression (GTEx) Project (analysis date:
   January 19, 2018) and found that p73 is expressed at 0.6 transcripts
   per million (TPM). Specifically, TAp73α and TAp73β are the predominant
   N-terminal isoforms expressed in human ovaries ([79]Figure S1D).

   Given the potential for hetero-oligomerization between p73 and its
   family member p63 ([80]Chan et al., 2004, [81]Rocco et al., 2006,
   [82]Harms and Chen, 2006), we determined if p63 and p73 were
   co-expressed during follicular development using dual IF detection of
   the proteins. p63 expression was restricted to oocytes (arrowhead) of
   primordial follicles in p73+/+ ([83]Figure S1E, top panel), consistent
   with previously published data showing that p63 expression in
   primordial follicles promotes genome integrity during meiotic arrest
   ([84]Suh et al., 2006). The oocyte-restricted expression pattern of p63
   was unaltered in p73−/− ovaries ([85]Figure S1E, lower panel).

   The ovarian phenotypes observed in our p73−/− female mice are
   consistent with the fertility defects reported in TAp73-deficient
   animals, but not ΔNp73-deficient mice, given that both females and
   males are fertile in the latter ([86]Tomasini et al., 2008, [87]Wilhelm
   et al., 2010). TAp73-deficient females showed defective follicle
   development and significantly decreased ovulation rate relative to
   wild-type animals in response to exogenous hormone stimulus, and the
   few ovulated oocytes observed were trapped under the bursa and unable
   to reach the fallopian tube for implantation ([88]Tomasini et al.,
   2008), possibly due to the lack of ciliated cells lining the oviduct
   ([89]Marshall et al., 2016). The corpora lutea are the primary sites of
   progesterone production after ovulation in mice ([90]Allen, 1941,
   [91]Rothchild, 1981). Therefore, the lack of corpora lutea observed in
   our p73−/− female mice ([92]Figure 1B) led us to compare the levels of
   circulating hormones in the p73+/+ and p73−/− female mice.

Loss of p73 Leads to a Significant Decrease in Circulating Progesterone

   Given the impact of p73 loss on folliculogenesis and the number of
   corpora lutea, we measured circulating progesterone in nulliparous
   p73+/+ and p73−/− mice at 6, 9, and 12 weeks of age. Progesterone
   levels fluctuate throughout the stages of the estrous cycle ([93]Byers
   et al., 2012). We assessed the estrous cycle through vaginal cytology
   and observed that p73−/− mice exhibit aberrant estrous cycle with
   prolonged diestrus stage compared with p73+/+ ([94]Figure S2A). Given
   the acyclic nature of p73−/− female mice, all circulating hormones were
   analyzed from the only shared stage (diestrus) between both phenotypes.
   Progesterone levels decreased nearly 75% in p73−/− samples collected at
   6 weeks of age (p value < 0.05) and 82% (p value < 0.01) at 12 weeks of
   age relative to p73+/+ mice ([95]Figure 2A). Circulating estradiol
   levels in p73−/− mice were similar to those in p73+/+ mice at each time
   point analyzed ([96]Figure 2B), although testosterone was reduced 55%
   in p73−/− samples harvested at 12 weeks of age (p value <0.01)
   ([97]Figure 2C).

Figure 2.

   [98]Figure 2
   [99]Open in a new tab

   Analysis of Circulating Progesterone in p73+/+ and p73−/− Female Mice

   Plasma levels measure by ELISA of (A) progesterone, (B) estradiol, and
   (C) testosterone from five female mice per genotype at 6, 9, and
   12 weeks of age; assay sensitivity range 0.2–50 ng/mL, 1–100 pg/mL, and
   5–100 pg/mL, respectively. Bars represent the mean. *p value < 0.05,
   **p value < 0.01.

   See also [100]Figure S2.

   Circulating hormones originating from the pituitary gland, including
   follicle-stimulating hormone (FSH), luteinizing hormone (LH), and
   growth hormone (GH), are key regulators of the estrous cycle and thus
   could exert a marked influence in ovarian progesterone production.
   Therefore, we measured circulating FSH levels in 12-week-old
   nulliparous mice, finding a 50% decrease in FSH levels in p73−/−
   compared with age-matched p73+/+ mice (p value <0.01)
   ([101]Figure S2B). These findings are consistent with previous studies
   showing that FSH promotes granulosa-theca cell interactions that drive
   the production of ovarian testosterone ([102]Smyth et al., 1993), thus
   constituting a possible mechanism to explain the decrease in
   testosterone observed in p73−/− female mice. LH levels were modestly
   decreased in p73−/− mice, albeit not to a statistically significant
   degree ([103]Figure S2C), and no difference in circulating GH was
   observed between p73+/+ and p73−/− samples ([104]Figure S2D).

   To determine if the hormonal differences in the p73−/− mice were linked
   to gross abnormalities in pituitary gland morphology due to hippocampal
   dysgenesis and hydrocephalus ([105]Yang et al., 2000, [106]Talos
   et al., 2010), we analyzed pituitary gland tissue sections from p73+/+
   and p73−/− female mice. The murine pituitary gland is composed of
   anterior (pars distalis, D; and intermedia, I) and posterior (pars
   nervosa, N) lobes. Despite diminished FSH production in p73−/− mice, we
   did not observe any overt histological differences in the pars distalis
   (asterisk) ([107]Figure S2E), where gonadotropin hormones (FSH, LH, and
   GH) are produced and secreted. In addition, we evaluated p73 expression
   in the pituitary gland of p73+/+ mice through IF. We observed
   p73-positive cells in pars intermedia (solid-line box), but not in pars
   distalis (dashed-line box) ([108]Figure S2F). Previous studies have
   shown that TAp73-deficient male mice have normal levels of FSH, LH, and
   gonadotropin-releasing hormone ([109]Holembowski et al., 2014). In
   addition, immature TAp73-deficient ovaries fail to respond to exogenous
   hormone stimulation and show a significantly decreased ovulation rate
   compared with age-matched wild-type mice ([110]Tomasini et al., 2008),
   further supporting normal pituitary gland signaling. Granulosa cells in
   growing follicles produce activin and inhibin as a feedback mechanism
   to regulate FSH secretion ([111]Knight and Glister, 2006). Thus it is
   possible that the loss of p73 in granulosa cells, and the decreased
   number of growing follicles observed in p73−/− females, may negatively
   interfere with ovarian-pituitary feedback signaling required for proper
   FSH secretion.

Ectopic Progesterone Partially Rescues Ovarian Follicle Development in
p73-Deficient Mice

   To determine if ectopic progesterone could rescue ovarian follicle
   maturation, 60-day-release progesterone pellets (15 mg) were implanted
   subcutaneously in 5-week-old nulliparous p73+/+ and p73−/− female mice
   and ovaries were analyzed 21 days after implantation. The ovaries from
   p73−/− mice with the placebo pellet harbored fewer primary, secondary,
   and antral follicles and were substantially smaller than
   placebo-treated p73+/+ mice ([112]Figures 3A and 3B). However, ovaries
   from p73−/− mice treated with ectopic progesterone were similar in size
   to ovaries from placebo- or progesterone-treated p73+/+ mice
   ([113]Figure 3A) and contained a greater number of primary, secondary,
   and antral follicles than placebo-treated p73−/− ovaries, similar to
   what was seen in progesterone- or placebo-treated p73+/+ mice
   ([114]Figure 3C). As a control, circulating progesterone was measured
   at the time when the mice were killed to confirm that ectopic
   progesterone pellet increased circulating progesterone in p73−/− mice
   to levels comparable with both placebo- and progesterone-treated p73+/+
   mice ([115]Figure 3D).

Figure 3.

   [116]Figure 3
   [117]Open in a new tab

   Progesterone Rescues Follicle Development in p73-Deficient Ovaries

   Progesterone slow-release pellet or placebo control (15 mg/pellet,
   60-day extended release) were implanted in five female mice per
   genotype at 5 weeks of age.

   (A) Representative H&E images of placebo control and ectopic
   progesterone-treated ovaries of p73+/+ and p73−/− mice (scale bar,
   200 μm).

   (B and C) (B) Follicle quantification of placebo control and (C)
   progesterone-treated p73+/+ and p73−/− ovaries, respectively. Data are
   shown as number of follicles per ovary; Prd, primordial; Prm, primary;
   Sec, secondary; Ant, antral.

   (D) Plasma levels of progesterone were measured through ELISA from
   placebo control and progesterone-treated p73+/+ and p73−/− female mice.
   *p value < 0.05.

   See also [118]Figure S3.

   To assess the effect of ectopic progesterone on female sterility in our
   mouse model ([119]Marshall et al., 2016), we determined the
   reproductive ability of placebo- or progesterone-treated p73+/+ and
   p73−/− female mice through mating trials. Twelve-week-old p73+/+ and
   p73−/− female mice were implanted with placebo or progesterone pellets
   and housed with p73+/+ males for a period of 14 days. At day 15,
   pregnancy status was determined and tissue was harvested for
   histological analysis. In p73+/+, two of two placebo-treated and two of
   three progesterone-treated female mice became pregnant, indicating that
   the level of ectopic progesterone administered did not inhibit their
   reproductive ability. In contrast, none of the five p73−/− mice, either
   placebo- (two mice) or progesterone-treated (three mice), became
   pregnant (data not shown). These results were not surprising given the
   lack of ovulation, corpus luteum formation, and ciliated cells in the
   p73-deficient mice ([120]Marshall et al., 2016). Ciliated cells are
   required for transport of the oocyte through the oviduct and to the
   uterus ([121]Critoph and Dennis, 1977, [122]Halbert et al., 1976).
   Also, we analyzed the ability of ectopic progesterone to rescue corpus
   luteum formation by implanting placebo or progesterone pellets (5 mg)
   in 12-week-old nulliparous p73+/+ and p73−/− female mice. After
   21 days, corpora lutea (arrowhead) were observed in p73+/+ with placebo
   or progesterone pellet. However, administration of ectopic progesterone
   in p73−/− mice was not able to rescue the formation of corpora lutea
   ([123]Figure S3, arrowhead).

Ectopic Progesterone Rescues Lobulo-Alveolar Budding in p73-Deficient Mice

   Mice that lack progesterone receptor exhibit defects in follicle
   rupture, causing impaired ovulation, absence of corpora lutea, and
   female infertility ([124]Lydon et al., 1995, [125]Lydon et al., 1996),
   similar to the phenotypes observed in our p73−/− female mice.
   Furthermore, impaired progesterone signaling leads to defects in proper
   mammary gland development by impeding the formation of lobulo-alveolar
   buds, small grape-like epithelial protrusions that, in the event of
   pregnancy, will generate the milk-producing alveoli ([126]Hennighausen
   and Robinson, 2005, [127]Bocchinfuso et al., 2000, [128]Lydon et al.,
   1995). Given the similarities between the progesterone
   receptor-deficient mice and our p73−/− female mice, we assessed the
   hormonally responsive function of the mammary glands from mice treated
   with slow-release progesterone pellets, as described above. Using
   whole-mount carmine staining to visualize the mammary epithelium, we
   noted a 50% reduction (p value <0.01) in lobulo-alveolar budding in
   placebo-treated p73−/− mice compared with p73+/+ mice ([129]Figures 4A
   and 4B). Progesterone-treated p73−/− female mice exhibited a complete
   rescue of lobular-alveolar budding and comparable mammary gland
   morphology to progesterone-treated p73+/+ control ([130]Figure 4A).
   Ectopic progesterone significantly increased the number of
   lobulo-alveolar buds in p73−/− mammary gland by 200% (p value <0.001)
   compared with the mammary glands of placebo-treated p73−/− mice
   ([131]Figure 4B).

Figure 4.

   [132]Figure 4
   [133]Open in a new tab

   Ectopic Progesterone Rescues Lobulo-Alveolar Budding Defect in
   p73-Deficient Mammary Gland

   (A) Whole mammary mount stained with carmine alum shows that ectopic
   progesterone rescues lobular-alveolar budding in p73−/− female mice
   (scale bar, 200 mm).

   (B) Lobulo-alveolar budding quantification of placebo control or
   progesterone from p73+/+ and p73−/−; values shown represent the average
   number of side branches per primary branch. **p value< 0.01, ***p
   value < 0.001.

   See also [134]Figure S4.

   Of note, the average number of lobular-alveolar buds was reduced in
   p73−/− mice by 45% at 6 weeks (p value <0.05), 60% at 9 weeks (p value
   <0.001), and 66% at 12 weeks (p value <0.001) of age compared with
   p73+/+ littermates ([135]Figures S4A and S4B). Despite decreased
   lobulo-alveolar budding, the histological architecture of mammary
   glands was unaffected by loss of p73 ([136]Figure S4C), with a
   well-organized luminal cell layer displaying apico-basal polarization
   (asterisks) and basally oriented myoepithelial cell layer (arrowheads),
   similar to what was seen in the mammary ductal epithelium of p73+/+
   littermates. IF staining in p73+/+ mammary glands revealed nuclear p73
   localization in basally located cells that stained positive for the
   myoepithelial/basal cell marker keratin 14 ([137]Figure S4C). As
   expected, we did not observe p73 expression in p73−/− mammary glands
   ([138]Figure S4C). Similar to our analyses in the ovaries, IF staining
   of p73+/+ mammary glands with antibodies against p63 confirmed
   myoepithelial/basal localization of p63 and p73 ([139]Figure S4D)
   ([140]Yang et al., 1999, [141]Barbareschi et al., 2001). Interestingly,
   p63 co-localized with a subset of p73-expressing cells in the
   myoepithelial layer suggesting that these two proteins interact in
   specific myoepithelial cells of the mammary gland. Furthermore, we
   found that p63 expression was retained in the myoepithelial layer of
   p73−/− mammary glands, suggesting that decreased lobulo-alveolar
   budding in p73−/− is not due to impaired p63 expression
   ([142]Figure S4D).

p73 Regulates a Biological Adhesion Gene Network in Murine Granulosa Cells

   To gain mechanistic insight into the defects in follicle development
   and ovulation in p73−/− ovaries, laser capture microdissection was used
   to isolate granulosa cells from p73+/+ and p73−/− antral follicles of
   age-matched nulliparous mice (three mice per genotype). Principal
   component analysis revealed a clear separation between p73+/+ and
   p73−/− transcriptional changes in antral follicle samples in principal
   component 1 ([143]Figure 5A). Accordingly, we identified 3,209
   protein-coding genes differentially expressed between p73+/+ and p73−/−
   antral follicles ([144]Table S1), of which 1,603 were enriched in
   p73+/+ antral follicles and 1,606 were enriched in p73−/− antral
   follicles. Gene Ontology (GO) enrichment (maximum false discovery rate
   [FDR < 2.22 × 10^−16]) identified nine GO categories related to
   biological adhesion, including extracellular matrix organization,
   positive regulation of cell adhesion, cell-substrate adhesion, positive
   regulation of locomotion, extracellular structure organization,
   positive regulation of cellular component movement, and positive
   regulation of cell motility ([145]Figure 5B; [146]Table S2). These
   findings are consistent with previous reports of p73-mediated
   regulation of cell-cell adhesion and migration through integrin-β4
   ([147]Xie et al., 2018), vascular endothelial growth factor , and
   transforming growth factor β signaling ([148]Fernandez-Alonso et al.,
   2015, [149]Martin-Lopez et al., 2017, [150]Bae et al., 2018).

Figure 5.

   [151]Figure 5
   [152]Open in a new tab

   p73 Regulates a Gene Network Involved in Biological Adhesions in Antral
   Follicles

   (A) Principal component analysis (PCA) plot of RNA sequencing (RNA-seq)
   analysis from LCM-isolated p73+/+ and p73−/− antral follicles (n = 3
   mice/genotype).

   (B) Table shows top nine GO categories enriched in p73+/+ versus p73−/−
   antral follicles (FDR < 2.22 × 10^−16).

   (C) PCA plot of RNA-seq analysis from p73+/+ and p73−/− MGCs after
   ectopic p73 or control.

   (D) Venn diagram showing the overlap between genes expressed in p73+/+
   antral follicles (TPM > 1) and upregulated after ectopic p73 expression
   in p73+/+ and p73−/− MGCs.

   (E) Table listing the top 19 GO categories enriched in 1,649
   overlapping genes from (D).

   (F) Heatmap of expression for core 208 p73-upregulated granulosa cell
   genes. These genes were selected by identifying the 1,649 genes in (D)
   that were present in three or more of the enriched GO categories from
   (E) (FDR p value < 0.1).

   See also [153]Figure S5 and [154]Tables S1, [155]S2, [156]S3, [157]S4,
   [158]S5 and [159]S6.

   We isolated and cultured primary mouse granulosa cells (MGCs) from
   p73+/+ mice and transduced the cells with lentivirus expressing TAp73β
   for 48 hr. We did not observe any difference in cell morphology or the
   levels of cleaved poly (ADP-ribose) polymerase 1 (PARP1) between
   control MGCs and those expressing ectopic p73 ([160]Figures S5A and
   S5B). The rationale for using TAp73β was based on previously published
   data showing that TAp73β exhibits the highest level of transcriptional
   activity among p73 isoforms ([161]Ueda et al., 1999, [162]Lee and La
   Thangue, 1999) as well as the fact that the TAp73β isoform is highly
   expressed in human ovaries ([163]Figure S1D). We measured global gene
   expression changes by RNA sequencing after ectopic p73 in MGCs isolated
   from both p73+/+ and p73−/− female mice and identified clear separation
   of samples after ectopic p73 ([164]Figure 5C). Differential expression
   of 5,178 genes was identified in p73+/+ MGCs after ectopic expression
   of TAp73β, including 2,896 upregulated genes ([165]Table S3).
   Similarly, we identified 3,391 differentially expressed genes after
   TAp73β expression in p73−/− MGCs (2,087 upregulated genes) ([166]Table
   S4). We identified 1,649 genes commonly upregulated in
   TAp73β-expressing MGCs and in p73+/+ antral follicles (>1 TPM)
   ([167]Figure 5D, [168]Table S5). GO pathway enrichment analysis
   identified 19 GO categories (FDR < 2.22 × 10^−16), including biological
   adhesion and migration ([169]Figure 5E and [170]Table S6). A core set
   of 208 genes overlapped in at least 3 of the 19 enriched GO categories
   ([171]Figures 5F and [172]S5C), including Adam10, Adamts12, Icam1, Pxn,
   and Mmp14. Importantly, we identified multiple genes required for the
   formation of the follicular focimatrix (focal intra-epithelial matrix),
   which is the extracellular matrix that aggregates between granulosa
   cells and increases as follicles progress to pre-ovulatory stage
   ([173]Irving-Rodgers et al., 2004, [174]Irving-Rodgers et al., 2009,
   [175]Irving-Rodgers et al., 2010). Key focimatrix genes identified
   include Lama1, Lama2, Lama5, Lamb1, Hspg2, Nid1, and Nid2. These data
   suggest that p73 is a key regulator of genes involved in cell adhesion
   and migration.

p73 Regulates Cell Adhesion and Migration

   We used a well-established in vitro migration assay to analyze the
   effect of p73 on cell adhesion and migration. To set up the assay,
   magnetically attachable stencils (MAtS) were placed in culture vessels
   to create “gaps” in monolayer cell cultures ([176]Ashby et al., 2012).
   The measurement of cell movement into the gaps over time provided a
   reporter assay for cell adhesion and migration. Due to technical
   constraints of large-scale MGC isolation and culture, we used mouse
   embryonic fibroblasts (MEFs) isolated from p73+/+ and p73−/− embryos as
   well as transformed epithelial cell lines that readily grow in culture.
   Lentivirally delivered short hairpin RNA sequences against p73 were
   used to “knockdown” p73 in transformed cell lines (MDA-MB-231 and
   HCC1806). Cells were plated near confluency surrounding the MAtS and
   cultured in serum-free media for 14 hr. MAtS were then removed (T =
   0 hr) and migration was monitored for 8–11 hr, revealing that loss of
   p73 significantly decreased cellular migration rate in p73−/− MEFs,
   MDA-MB-231, and HCC1806 cells when compared with p73+/+ MEFs or vector
   control ([177]Figure 6A). Loss of p73 expression was confirmed through
   qRT-PCR and western blot analysis ([178]Figures S6A and S6B).

Figure 6.

   [179]Figure 6
   [180]Open in a new tab

   p73 Regulates Cell Migration

   (A) MEFs isolated from p73+/+ and p73−/− mice, MDA-MB-231, and HCC1806
   cells stably expressing control short hairpin RNA (shRNA) and p73 shRNA
   were plated in culture dishes containing magnetic stencils and grown to
   confluency (scale bar, 100 μm). Each dot represents the percentage gap
   closure per field of view. ***p value < 0.001.

   (B) Table listing adhesion- and migration-associated genes from the
   core set of 208 genes bound by p73 within 25 kb of their TSS (in
   HCC1806 cells). For each gene, the q value of the nearby p73 peak and
   its distance from the TSS of the gene are included.

   (C) Integrative Genomics Viewer images for selected genes from (B) with
   tracks for input, p73, and Pol II ChIP-seq in HCC1806 cells. Each
   sample was normalized to 1X depth of coverage. Individual tracks within
   a gene are scaled equally. RefSeq gene annotations are in blue
   schematics at the bottom of each panel on the same scale as the
   ChIP-seq tracks.

   See also [181]Figures S6 and [182]S7 and [183]Table S7.

   Given that the most significant difference in cell migration was
   observed in p73-deficient HCC1806 cells, we performed chromatin
   immunoprecipitation sequencing (ChIP-seq) on parental HCC1806 cells to
   determine if p73 directly binds near the transcriptional start site
   (TSS) of genes involved in cell migration. HCC1806 cells were
   formaldehyde cross-linked and processed for p73 and RNA polymerase II
   (Pol II) ChIP-seq, as described in the [184]Transparent Methods.
   Quality control analysis of these data demonstrated clear separation
   between ChIP and input signal for p73 and Pol II ([185]Figure S7A).
   Because the p73 ChIP replicates were highly correlated
   ([186]Figure S7B), we pooled the samples for peak calling to increase
   peak detection sensitivity. We identified 3,555 p73 and 19,696 Pol II
   genomic binding sites ([187]Table S7). Motif analysis showed strong
   enrichment for the p53 family binding motif ([188]Figure S7C)
   ([189]Rosenbluth et al., 2008, [190]el-Deiry et al., 1992, [191]Lokshin
   et al., 2007, [192]Smeenk et al., 2008). We identified known binding
   sites in p73 target genes MDM2 and CDKN1A ([193]Figures S7D and S7E)
   ([194]Barak et al., 1993, [195]Juven et al., 1993, [196]Espinosa and
   Emerson, 2001) ([197]Robinson et al., 2011, [198]Thorvaldsdottir
   et al., 2013) as well as a binding site in the newly reported p73
   target gene ITGB4 (integrin-β4) ([199]Xie et al., 2018). Since we were
   comparing murine gene expression data with human ChIP data, we focused
   our analysis on genes that were increased after p73 expression in MGCS
   and for which the binding of p73 occurred within 25 kb of the TSS in
   HCC1806 ChIP. From the 208 p73-regulated core gene set, we found 30
   adhesion- and migration-associated genes with a p73 binding site within
   25 kb of the TSS of the human gene homolog ([200]Figure 6B). Of
   immediate interest were p73 binding sites near genes encoding adhesion
   PXN and focimatrix components LAMA5, NID1, and NID2 ([201]Figure 6C).
   Paxillin is a scaffolding protein that regulates cytoskeleton
   remodeling, cell migration, and focal adhesions ([202]Huang et al.,
   2003, [203]Hu et al., 2014, [204]Deramaudt et al., 2014).

   p73 is necessary for cell migration in transformed epithelial cell line
   models. Through ChIP-seq, we identified p73 binding within 25 kb of the
   TSS of genes involved in cell-to-cell adhesion and migration, including
   NID1, NID2, LAMA5, and PXN. Furthermore, ectopic p73 expression is
   sufficient to upregulate the expression of these genes in MGCs.
   Collectively, these data support the conclusion that p73 regulates
   ovarian folliculogenesis and ovulation, in part through regulated
   expression of adhesion and focimatrix genes necessary for proper
   follicle maturation.

Discussion

   We discovered that p73 is required for proper ovarian follicle
   development, ovulation, and subsequent corpus luteum formation and
   progesterone production. Similar to prior findings made with
   TA-specific p73-deficient male mice ([205]Inoue et al., 2014), we
   observed a significant decrease in levels of circulating progesterone
   in our p73−/− female mice. Furthermore, we demonstrated that the
   mammary branching defect observed in p73−/− female mice is secondary to
   decreased levels of progesterone through a complete rescue of the
   branching defect after ectopic progesterone administration. Through
   analysis of gene expression between p73+/+ and p73−/− antral follicles
   and modulation of p73 expression in various model systems, from primary
   culture of MGCs to human epithelial cells, we discovered p73-dependent
   regulation of genes crucial for biological adhesion (Pxn) and
   extracellular matrix interactions required to form proper focimatrix
   (Lama5, Nid1/2, and Hspg2). Focimatrix levels have been previously
   linked with steroidogenesis and CYP11A1 ([206]Irving-Rodgers et al.,
   2009, [207]Matti et al., 2010) during ovarian follicle development.
   Consistent with defective focimatrix formation in our p73-deficient
   ovarian follicles, we observed a significant decrease in expression of
   Cyp11a1, as well as other hormonally regulated genes including
   prolactin receptor (Prlr), luteinizing hormone/choriogonadotropin
   receptor (Lhcgr), oxytocin receptor (Oxtr), steroidogenic factor 1
   (Nr5a1), and activin B receptor (Acvr1c) in p73−/− antral follicles
   ([208]Table S1). NR5A1 is a transcriptional activator required for the
   formation of steroidogenic tissues, and cell-specific knockout
   experiments have shown that Nr5a1 is necessary for male and female
   fertility ([209]Ferraz-de-Souza et al., 2011, [210]Jeyasuria et al.,
   2004). Mice that lack ACVR1C expression in granulosa cells exhibit
   striking similarities to our p73−/− mice including defective follicle
   development, absence of corpora lutea, and decreased levels of
   circulating FSH ([211]Sandoval-Guzman et al., 2012), providing a
   possible mechanism for the decreased FSH levels in our p73−/− females.
   Future studies are needed to determine the direct or indirect mechanism
   by which p73 regulates the expression of genes required for proper
   steroidogenesis and hormone signaling in antral follicles.

   The lack of functional p73 protein in murine ovaries results in an
   absence of corpora lutea and an increase in the number of primordial
   follicles, suggesting a defect in primordial-to-primary follicle
   transition. We also observed a decrease in FSH levels, which supports
   the reduced number of developing follicles in p73−/− mice. FSH,
   secreted from the pituitary gland, is positively and negatively
   regulated by activin and inhibin, respectively, which are secreted from
   granulosa cells ([212]Knight and Glister, 2006). From our analysis, p73
   is expressed in the pars intermedia, and not in pars distalis where
   FSH, LH, and GH are produced. Previous studies have demonstrated that
   p73-deficient mice exhibit hippocampal dysgenesis and hydrocephalus
   ([213]Yang et al., 2000, [214]Talos et al., 2010, [215]Marshall et al.,
   2016). In our p73−/− mice, we also observed hippocampal dysgenesis and
   hydrocephalus to varying degrees and on a mouse-to-mouse basis across
   our cohort of p73-deficient mice; we are not able to rule out the
   possible effect of these phenotypes on pituitary gland function. Future
   experiments are needed to determine the impact of hippocampal
   dysgenesis and hydrocephalus on pituitary gland signaling and hormone
   secretion.

   p73 is necessary for multiciliated cell development ([216]Marshall
   et al., 2016, [217]Nemajerova et al., 2016), and we observed expression
   of p73 in ciliated cells that line the oviductal epithelium in mice
   ([218]Marshall et al., 2016). A major role of these ciliated cells is
   to transport the oocyte to the uterus for implantation ([219]Critoph
   and Dennis, 1977, [220]Halbert et al., 1976). Mouse models that lack
   ciliated cells, such as gene knockouts of Foxj1 ([221]Gomperts et al.,
   2004) and Gemc1 ([222]Terre et al., 2016), have similar phenotypes to
   our p73-deficient mice ([223]Marshall et al., 2016), including female
   and male infertility. Gemc1−/− ovaries exhibit a follicle development
   defect similar to our p73−/− female mice ([224]Terre et al., 2016). We
   are not able to rule out a possible paracrine mechanism between the
   ciliated cells in the oviduct and the granulosa cells in the ovaries.
   It would be of interest to determine if ciliated cells have additional
   roles in fertility besides transport of the oocyte, in terms of a
   direct effect on ovarian follicle development and ovulation through
   cell-to-cell signaling mechanisms. We posit that p73 is required in
   both granulosa cells within the follicle and ciliated cells in the
   oviduct to promote proper follicle development, ovulation, and oocyte
   implantation.

   In summary, we discovered that p73 is required for ovarian follicle
   development, ovulation, and subsequent corpus luteum formation. The
   mammary gland branching defect observed in our p73−/− female mice was
   due to decreased circulating levels of progesterone. We identified a
   p73-regulated core gene set involved in biological adhesion and cell
   migration in MGCs. Furthermore, we demonstrated that p73 is necessary
   for proper epithelial cell migration and identified p73 binding near
   the TSS of focimatrix component genes and crucial regulators of cell
   adhesion and migration like PXN. We conclude that p73 acts as critical
   regulator of cell-to-cell adhesion, extracellular matrix interactions,
   and cell migration and promotes proper follicle development, ovulation,
   and fertility.

Limitations of the Study

   A limitation of the study was our inability to isolate and culture
   sufficient numbers of the murine granulosa cells required for cell
   migration and ChIP experiments, due to technical constraints associated
   with purification of cells from murine ovaries. To address this
   limitation, we used another tissue for generation of primary cultures
   of murine cells from p73+/+ and p73−/− mice as well as the established
   HCC1806 cell line to analyze the role of p73 in cell migration. For
   continuity, we used HCC1806 cell line to identify genes involved in
   migration, to which p73 directly binds through ChIP-seq.

Methods

   All methods can be found in the accompanying [225]Transparent Methods
   supplemental file.

Acknowledgments