Effect of the spatial–temporal specific theca cell Cyp17 overexpression on the reproductive phenotype of the novel TC17 mouse

In the ovarian follicle, the Theca Cells (TCs) have two main functions: preserving morphological integrity and, importantly, secreting steroid androgen hormones. TCs express the essential enzyme 17α-hydroxylase/17,20-desmolase (CYP17), which permits the conversion of pregnenolone and progesterone into androgens. Dysregulation of CYP17 enzyme activity due to an intrinsic ovarian defect is hypothesized to be a cause of hyperandrogenism in women. Androgen excess is observed in women with polycystic ovary syndrome (PCOS) resulting from excess endogenous androgen production, and in transgender males undergoing exogenous testosterone therapy after female sex assignment at birth. However, the molecular and morphological effects of Cyp17 overexpression and androgen excess on folliculogenesis is unknown. In this work, seeking a comprehensive profiling of the local outcomes of the androgen excess in the ovary, we generated a transgenic mouse model (TC17) with doxycycline (Dox)-induced Cyp17 overexpression in a local and temporal manner. TC17 mice were obtained by a combination of the Tet-dependent expression system and the Cre/LoxP gene control system. Ovaries of Dox-treated TC17 mice overexpressed Cyp17 specifically in TCs, inducing high testosterone levels. Surprisingly, TC17 ovarian morphology resembled the human ovarian features of testosterone-treated transgender men (partially impaired folliculogenesis, hypertrophic or luteinized stromal cells, atretic follicles, and collapsed clusters). We additionally assessed TC17 fertility denoting a perturbation of the normal reproductive functions (e.g., low pregnancy rate and numbers of pups per litter). Finally, RNAseq analysis permitted us to identify dysregulated genes (Lhcgr, Fshr, Runx1) and pathways (Extra Cellular Matrix and Steroid Synthesis). Our novel mouse model is a versatile tool to provide innovative insights into study the effects of Cyp17 overexpression and hyperandrogenism in the ovary.


Introduction
functional roles of the theca layer are mainly achieved by two different cell types, an inner theca interna and an outer theca externa [8]. The terminal differentiation of the theca externa is characterized by expression of contractile proteins that play critical roles leading to follicle rupture in the ovulation process [9]. On the other hand, terminal differentiation of the theca interna is characterized by development of theca interstitial cells that produce androgens. Understanding this developmental process is important because theca-derived androgens are obligatory precursors for follicle estrogen production, and are essential for normal folliculogenesis, ovulation, and reproduction [10].
Focusing on TCs and their androgenic role in female reproduction has been conceptually overlooked, with androgens being primarily studied in the context of male reproductive function. However, in recent decades, novel cellular and molecular methodologies as well as animal models have revealed new insights into the impact of TC androgen production on follicle progression and the onset of ovarian disorders such as polycystic ovary syndrome (PCOS) [11][12][13][14][15][16][17][18]. Therefore, understanding the role of TCs and their androgenic steroids on follicle maturation has significantly evolved over time [19]. Initially, androgens were considered detrimental to normal folliculogenesis, mainly due to the negative effects demonstrated in mouse models [20][21][22][23]. The afterward evolving narrative, built on androgen receptor (AR) investigations [24,25], appeared to be more complex. Indeed, TCs were found to also promote crosstalk among GCs and oocytes during development and to support the growing follicle as it progresses through developmental stages to produce a mature oocyte [26][27][28]. In vitro studies have shown that androgen can promote GC differentiation, but this appeared to be stage dependent as with increased growth, larger size follicles may become less responsive and even atretic in response to androgen exposure [13,17,29]. To date, it is known that androgens are important for normal ovarian follicle maturation since small follicle growth is enhanced by androgens [13,17,[29][30][31]. It has been hypothesized that this effect might be the result of the ability of androgens to increase FSH receptor in GCs [14,15].
Notably, steroidogenic TCs uniquely express the essential enzyme 17α-hydroxylase/17,20-desmolase (CYP17), which is required for androgen production [7,[32][33][34]. In the female mouse, Cyp17 expression is primarily restricted to the ovary (~ 500 transcripts per million, TPM) and placenta, with faint expression (~ 2 TPM) in the uterus and adrenals. Within ovarian follicles, Cyp17 is expressed in TCs but not in adjacent GCs or in oocytes [35,36]. Most importantly in women with PCOS, androgen overproduction likely results, at least in part, from dysregulation of Cyp17 enzyme activity due to an intrinsic defect of the TCs [37][38][39]. This is supported by studies demonstrating elevated levels of Cyp17 mRNA and protein expression in TCs of ovaries from women with this disorder [30,40]. However, most of these studies were performed in PCOS patients and, therefore, are associated with intrinsic morphological and functional ovarian defects that cannot recapitulate the genuine role of TCs in the normal ovary. Therefore, the physiological role of androgens on follicle function remains unclear. This limitation is not trivial since comprehensive knowledge of the effects of androgens on ovarian function in normal women is very limited. The closest experimental evidence, appropriately focused on the androgens effect in non-pathological ovaries, have been transgender male (TGM) studies which were unfortunately characterized by limited power and lack conclusive results [41][42][43].
As a result, there is an absence of reliable information regarding the effect of androgen on normal follicle function. To address these gaps in knowledge, we created, by a combination of the Cre/LoxP and the Tet-dependent (on-off switch) expression systems, a transgenic mouse model inducibly overexpressing Cyp17, which we called TC17. This strategy differs from other animal models of androgen excess that have involved in vivo and systemic administration of a single androgen or aromatase inhibitor (e.g., Letrozole) [44][45][46]. Remarkably, our TC17 recapitulated the ovarian morphology observed in TGM treated with gender affirming testosterone therapy and appears to be a valuable model to study the ovarian folliculogenesis in presence of local long term androgen excess.

Plasmids and mouse models
All mice were C57BL/6 J (B6) background (Jackson or Envigo, USA). We generated a breeding line of mice overexpressing TC-selective Cyp17 using a combination of the Tet-dependent expression system and the Cre/LoxP gene control system as outlined in Fig. 1B. The combination of Tet-based induction and Cre/LoxP gene control is a newer system developed to produce transgenic animal models to study the molecular basis of human disease in adult animals in a temporal manner. This elegant strategy is widely used in vivo and in vitro for conditional, reversible gene expression [47][48][49][50][51][52][53][54][55][56][57][58][59]. Specifically, we have used Cyp17 promoter-iCre mice [60] crossed with transactivator mice (R26-STOP-rtTA-IRES-EGFP transgene at the ROSA26 locus, Jackson Lab) and with responder mice carrying the TRE-Cyp17 transgene created at the University of California, San Diego (UCSD) transgenic mouse and embryonic stem cell core facility. The Cyp17 coding segment was inserted into the multi-cloning site of the TRE-PminCMV vector (pTRE-TightTM, Clontech, Mountain View, CA) which contains a modified Tet responsive element (TRE) that is silent in the absence of rtTA and Doxycycline (Dox) treatment. A two-step breeding process was used to obtain transgenic mice. Initially, we paired transactivator and responder mice to produce double transgenic mice (R26-STOP-rtTA-IRES-EGFP/TRE-Cyp17). Subsequently, we mated the double transgenic mice with iCre-expressing mice to get the experimental tri-transgenic mice (R26-STOP-rtTA-IRES-EGFP/TRE-Cyp17/Cyp17iCre). Here, Cyp17 promoter-iCre mice [60] were used to ensure rtTA/ EGFP was expressed specifically in TCs of secondary follicles. Importantly, after the DNA segment between the two LoxP sites was excised by Cyp17iCre specifically in TCs, the R26-STOP-rtTA gene remained activated in all daughter TCs. Double transgenic mice with WT Cyp17 gene (R26-STOP-rtTA-IRES-EGFP/Cyp17iCre) and double transgenic mice without rtTA/TetOn gene (TRE-Cyp17/Cyp17iCre were considered control mice (CTRL) for the study. Only upon treatment with Dox can suppression be relieved and active transcription of TRE-Cyp17 be induced. Pilot studies were performed to validate the model (Fig. 1A, Additional file 1: Figure  S1, Additional file 1: Figure S2). Progressive administration of intraperitoneal (i.p.) injected Dox exerted a dosedependent induced gene expression increase in vivo in transgenic mice (Additional file 1: Figure S1). A pilot in vitro study was performed to validate the efficiency of the systems (Fig. 1A). Mice were group-housed in a

Tissue collection and histology
After Dox chow exposure, mice were anesthetized with isoflurane, weighed, and blood was collected via retroorbital bleeding before rapid euthanizing. After extraction, ovaries were weighed. One ovary from each mouse was collected, frozen on dry ice, and stored at − 80 °C until processing for mRNA expression levels using quantitative PCR. The other ovary from each mouse was fixed in 10% formalin at 4 °C overnight and stored in 70% ethanol before histologic processing. For histological analysis, fixed ovaries were serially sectioned at 10 μm and then stained with hematoxylin and eosin (H&E) by the UCSD Tissue Technology Shared Resource (formerly known as Histology and Immunohistochemistry Core). Primary, secondary, antral, and cystic follicles were counted from two sections randomly selected from each ovary. In each case, counts were made by one investigator blind to the treatment group.

Hormone assays
LH, FSH, and E2 hormone levels were measured by the University of Virginia Ligand Core Facility. Serum LH and FSH were measured by a mouse multiplex assay (reportable range 0.24-30.0 ng/ml and 2.4-300 ng/ml, respectively). Serum E2 was measured using a mouse enzyme-linked immunosorbent assay (range 3.0-300 pg/ ml). Serum T was measured with LC-MS/MS at the UCSD Health Center for Advanced Laboratory Medicine (range 4-1560 ng/dL).

Estrous cycle and fertility assessment
Estrous cycles were monitored over a period of 15 days by light microscopic analysis of the predominant cell type in vaginal epithelial smears obtained 4-6 weeks after Dox or control chow treatment. Proestrus was categorized by the presence of nucleated and some cornified epithelial cells, estrus by the presence of cornified cells, and metestrus/diestrus for the presence of some cornified epithelial cells and primarily leukocytes. A separate cohort of female TC17 and CTRL mice at 7 weeks old was used to assess fertility. At the age of 8 weeks and 1 week after treatment (n = 10/group), TC17 were paired with adult C57BL/6 N breeder males at 3 months old. Breeder males were removed after 10 days, and females were assessed for pregnancy, time to first litter, and number of pups per litter.

Hematological profile
After collection of blood, Hemavet 950FS was used to obtain the hematological profile of TC17 and CTRL mice. The control ranges for RBC (M/μl) and for HCT (%) were 6.36-9.42 and 35.1-45.4, respectively.

Transient transfections
293 T cells were cultured into 6-well tissue culture plates (1 × 10 6 cells/well). The medium was replaced the day after with serum free DMEM-F12 (complemented with antibiotics), and transfection was performed using the above-described plasmids for 4 h with Lipofectamine LTX with Plus or Lipofectamine 3000 reagents (catalog #15338100 or #L3000008, respectively, Thermo Fisher Scientific) following manufacturer's procedures. After 4 h, the medium was replaced with new serum free DMEM-F12 and transfected cells were cultured at 37 °C for 24 h with or without Dox. At this point, cells were lysed for further analyses.

RNA extractions and q-RT-PCR
After 4-h transfection and additional culture for 24 h in serum free DMEM-F12, 293 T and total ovaries were lysed using TRIzol reagent (catalog #15596026, Thermo  2). Read quantification was called using FeatureCounts, and differential expression analysis was called using DESeq2, with an adjusted p-value threshold of 0.05. Additionally, GO and KEGG enrichment analyses were performed using Clus-terProfiler using an adjusted p-value threshold of 0.05 [61][62][63].

Confocal and RNAscope
Mice were deeply anesthetized with Medetomidine/Ketamine. When no response to tail/toe pinch was present, mice were transcardially perfused with 1% phosphatebuffered saline solution first, followed by 4% paraformaldehyde solution to fix the ovary tissue. The ovaries were then removed from the mice and kept in a 30% sucrose solution until use. Frozen ovaries were sectioned (30 μm

Dox treatment in TC17 transgenic mice induced expression of Cyp17 in a local and temporal manner and increased Testosterone blood levels
After in vitro validation of the trans genetic constructs used in the present work (Fig. 1A) and the execution of breeding strategy to obtain TC17 mice (see Material and Methods and Fig. 1B), the novel TC17 model was doxtreated by i.p. injection for a dose-response (Additional file 1: Figure S1). The expression of Cyp17 appeared to be dose dependent. To validate the effective long-term upregulation of Cyp17, we used Dox diet as a more convenient administration method. After long-term treatment (4 weeks) mice were euthanized, and Cyp17 upregulation was validated by RNAscope and qPCR (Fig. 2). Strikingly, RNAscope analysis of the TC17 ovaries clearly showed specific upregulation of Cyp17 in TCs compared with the CTRL (Fig. 2A). Increased Cyp17 mRNA levels were analyzed and confirmed by qPCR (Fig. 2B). Also, the system specifically expressed the transactivator rtTA protein in the TC17 TCs as shown by microscopy (Additional file 1: Fig. 2).
To further validate our model, we quantified hormone levels in the serum. As expected, T was strongly  N = 4). RNAscope was performed for Cyp17 probe and Draq5 to stain DNA. Representative confocal micrographs of CTRL (upper panel) and TC17 transgenic mouse ovaries (lower panel). Panels show the effects of Dox treatment in the Cyp17 expression (in red). An antisense control probe was used as control from the vendor. B qPCR validation of Cyp17 expression in TC17 ovaries compared with CTRL ovaries (N = 6). After Dox treatment (8 weeks), ovaries were collected, and qPCR was performed Graphs show fold change means ± s.e.m relative expression to Cyp17 following normalization to the housekeeping gene. Data were analyzed using the two-tailed Mann-Whitney test (***p < 0.001) upregulated in TC17 compared to CTRL (Fig. 3A). E2, FSH, and LH did not show significant differences (Fig. 3B-D).

TC17 ovarian phenotype was marked by impaired folliculogenesis, hypertrophic luteinized stomal cells, follicle atresia, and collapsed cell clusters
To further understand the long-term effect of the induced Cyp17 upregulation, female TC17 mice were treated with Dox for 8 weeks (Fig. 4). The schematic execution of the experiments is depicted in Fig. 4A. After treatment, TC17 body mass and ovarian weight were significantly higher compared to the CTRL mice (Fig. 4C). Histological assessment of the ovaries showed that TC17 presented a different morphology compared with the control with a high presence of stromal cells (Fig. 4B). TC17 ovaries were also characterized by impaired folliculogenesis with a significantly lower number of antral follicles (Fig. 5A-D and G) and hypertrophic stromal or luteinized stromal cells ( Fig. 5F and G). Moreover, atretic follicles and atretic cystic formations were observed (Fig. 5E). We also found morphological structures that relate to the luteinization of stroma rather than corpora. These structures (Fig. 5G)-which we called collapsed clusters-were composed of pools of lucent cells not discernable from the surrounding stroma.

TC17 female mice have delayed estrous cycle and impaired fertility
The evidence that TC17 ovaries can resemble that observed in transgender men creates a potential model for studying the effects of long-term hyperandrogenemia on the reproductive physiology of these individuals. As the first approach, we assessed the TC17 estrous cycle. At 5 weeks after treatment, TC17 female mice spent their estrous cycle in diestrus, according to the finding that leukocytes were predominant in their vaginal smears (Fig. 6A) and presented partial oligo-anovulation with an evident delay of the estrous (Fig. 6B). To assess functional consequences of the upregulation of Cyp17 and ovarian hyperandrogenism on reproduction, we evaluated fertility in TC17 and CTRL females beginning 1 week after Dox treatment (scheme of the experiment  Fig. 6C). All the CTRL females (10/10, 100%) gave birth to a litter following pairing with a breeder male for 10 days (Fig. 6D). In contrast, only two of the TC17 females (2/10, 20%) successfully gave birth. Signs of impaired fertility were also evident by significant differences in the time to birth the first litter (Fig. 6E) and litter size (Fig. 6F) compared to the CTRL group.

Transcriptional profiling of TC17 ovaries reveals genome-wide impact of Cyp17 upregulation
To investigate the transcriptional changes associated with the Dox-induced Cyp17 upregulation, and thus the effect of excess androgen, TC17 and CTRL ovaries were profiled using RNA sequencing (Fig. 7A). We then performed a differential expression analysis between CTRL and TC17 samples to identify genes induces or repressed by Cyp17 overexpression (Additional file 2: Table S1).
Next, to evaluate the functional significance of the genes found to be dysregulated in the TC17 model, we performed Gene Ontology (GO) analysis on DEGs. We found DEGs (Additional file 1: Figure S4) were significantly enriched in the extracellular matrix (ECM), collagen-containing ECM, and steroid metabolic process pathways (Fig. 7C, Additional file 1: Figure S5). These findings can help explain our histopathological findings, in which the main features were an increase of the stromatic component/luteinized tissue (see an increase of LH receptor and enriched GO Pathways associated with collagens and ECM) with specific transgender features and a partial impairment of the folliculogenesis (decreased Fshr levels).

TC17 mice have polycythemia
Finally, we sought to investigate if TC17 presented specific systemic signs associated with androgen overload. We found that TC17 blood was distinguished by polycythemia, with elevated Red Blood Cell (RBC) levels and hematocrit (HCT) percentage, as depicted in Additional file 1: Figure S7.

Discussion
In this work, we present a new transgenic mouse model, called TC17, which is characterized by the Dox-induced spatial and temporal Cyp17 upregulation in TCs. We generated responder mice with pTRE3G-Cyp17. By crossing these mice with transactivator mice (R26-STOP-rtTA-IRES-EGFP transgene at the ROSA26 locus) and iCre mice (Cyp17 promoter-iCre), we successfully obtained tri-transgenic mice overexpressing Dox-dependent Cyp17 in TCs of the ovary. Following long-term Dox treatment, Cyp17 mRNA levels from the ovaries of these TC17 mice revealed a Six to ten-fold increase compared with wild-type mouse ovaries. Consistent with overexpression of Cyp17, serum levels of T were significantly elevated with no significant change in E2, FSH, or LH. The endocrine profile of our model was also accompanied by significantly greater body and ovarian weight at the end of the treatment compared with controls. In addition, TC17 mice exhibited irregular estrous cycles and were characterized by reduced fertility, with a longer time to first litter and fewer pups per litter than wild-type mice. TC17 morphological ovarian assessment denoted partially impaired folliculogenesis with a significant decrease of antral follicles and hypertrophic stromal cells and increased presence of luteinized stromal cells. We also found high numbers of atretic/ Fig. 7 The transcriptomic effect of Cyp17 overexpression is the TC17 ovary. RNA sequencing reveals Cyp17 effect on ovary gene regulation. A Upon Cyp17 induction, 721 genes were upregulated, and 290 genes were downregulated (adj. p-value < 0.05). B Among these genes, well-known ovary morphogenesis transcriptional regulators such as Cyp11, Prlr were upregulated, while Ihh and Runx1 were downregulated. Volcano plot indicates the relative fold change and p-value of genes in B. C GO analysis of the 1011 differentially expressed genes included terms such as extracellular matrix and collagen-containing extracellular matrix (see Additional file 1: Figure S4 for full-term list). D-E The induction and repression of Lhcgr and Fshr respectively, as found by RNA-seq, were confirmed using qPCR cystic follicles and collapsed lucent cell clusters. Collectively, these data suggest an androgen-induced defect in normal folliculogenesis and fertility. Ovarian morphological features similar to those demonstrated in our TC17 model have been described in prior studies of Testosterone Replacement Therapy (TRT)-treated transgender men [43,[64][65][66][67][68]. Indeed, the TC17 mouse model appeared to resemble specifically several of these features: morphological ovarian assessment in denoted partially impaired folliculogenesis with a significant decrease of antral follicles. Moreover, hypertrophic stromal cells or luteinized stromal cells [69] similar to the ones observed in transgender man ovaries were detected [41,42,70,71]. Although we did not find polycystic ovarian morphology as described by Ikeda et al. we did observe high numbers of atretic/cystic follicles and collapsed lucent cell clusters described by the group [67].
To date, only one animal model has been proposed to investigate the impact of testosterone therapy on reproduction in transgender men. This model, by Kinnear et al. utilized subcutaneous administration of testosterone enanthate and mirrored several reproductive perturbations observed in transgender men on T therapy [43,72]. Interestingly, they showed that T therapy-induced interruption of estrous cyclicity is reversible [72]. However, pregnancy outcomes were not reported for this model, and did not demonstrate the ovarian hypertrophic stromal morphologies observed in humans.
Underlying the morphological changes induced by Cyp17 overexpression in our TC17 model were several molecular alterations. We found 1011 differentially expressed genes (290 down-and 721 upregulated) in ovaries from TC17 mice compared to those from CTRL mice. Among them, we found genes that can shed light on the ovarian histopathology we described. In the TC17 transcriptomic profile, genes controlling steroid synthesis (Star, Cyp11a1) were upregulated in the TC17 mice. The LH receptor gene (Lhcgr) was also significantly upregulated, explaining the high level of luteinized stromal cells. GO and KEGG analysis of these DEGs corroborated our hypothesis that TC17 can resemble the ovarian phenotype of testosterone-treated transgender men with enrichment of pathways for collagenization and the ECM organization.
Other important evidence of the TGM ovarian phenotype from our transcriptomic data included upregulation of the prolactin receptor (Prlr) gene and downregulation of the Runx1 and Foxl2 genes. The current literature indicates Prlr in the ovary has a luteotropic action [73]. Interestingly, Nicol et al. in 2019 found Runx1 essential for the maintenance of the ovary and the combined loss of Runx1 and Foxl2 partially masculinizes fetal ovaries [74]. TC17 was also characterized by polycythemia. High levels of HCT and RBCs are typically increased in TGM, and the subsequent polycythemia is considered an adverse drug reaction lifelong hormonal therapy [75,76].
Finally, in addition to the described molecular and morphological changes observed in the TC17 mice, impaired fertility was also observed. Our study uncovered that TC17 estrous cycles were disrupted, and pregnancy rates were significantly diminished. This is of particular importance given the lack of clinical data in humans describing the reproductive effects of gender-affirming TRT in TGM. Although TRT is the mainstay of gender-affirming medical care in TGM [77,78] and secondary amenorrhea is common in testosterone-treated individuals [79][80][81], the exact mechanism of menstrual suppression is unknown. Although one recent study observed high rates of anovulation in TGM undergoing TRT [82], few studies have assessed the effects of testosterone on ovarian follicle structure and function. Given this limited knowledge, the current standard of care is to counsel patients interested in gender-affirming testosterone therapy after female sex assignment at birth regarding the potential for decreased fertility [77,78,[83][84][85][86]. As the access to gender-affirming care improves, more patients are considering fertility preservation and its impact on their identity and future family goals [87]. As such, there is a critical need to perform a clinical investigation to carefully examine the effects of androgen therapy on normal ovulatory function.
Given the ethical difficulties of studying the reproductive consequences of high-dose testosterone therapy in humans, and the limitations imposed by cost, fecundity, generation time, and lifespan introduced when studying non-human primates, rodent models offer an attractive alternative.
Considering that, our investigations show that the novel TC17 model is an innovative and powerful tool for future investigations of the dose-dependent effects of androgen on ovarian structure and function, reproductive cyclicity, and fertility. In summary, TC17: (i) has a doxycycline-dependent regulation Cyp17 specifically in TCs, (ii) resembles TGM ovarian histopathology, (iii) mimics polycythemia condition which is typical in presence of hyperandrogenism (Fig. 8).