Leydig Research Articles

8030 Sexually Dimorphic Metabolic Consequences Of CCN5/WISP2 Gene Deletion Revealed A Male-Specific Pattern Of Gene Expression

Abstract Disclosure: M. Yang: None. V. Xega: None. M. Zakikhani: None. J. Liu: None. Matricellular protein CCN5/WISP2 is a novel target of IGF-1 and WNT signaling within the pancreatic islets. Our previous research suggests CCN5 promotes β-cell proliferation and survival through Akt/PKB and focal adhesion kinase signaling pathways. More recently, we have reported sexual dimorphic features of CCN5 gene deletion and overexpression. Specifically, male knockout mice demonstrated improved insulin sensitivity and glucose tolerance upon high-fat diet (HFD)-induced obesity, while females were unaffected (ENDO 2022). On the other hand, male aP2-CCN5 transgenic mice that overexpress CCN5 in adipose tissues were partially protected from HFD-streptozotocin induced diabetes, with improved glucose tolerance; females exhibited impaired glucose tolerance and exacerbated diabetes (ENDO 2023). Our findings indicate that an increased level of CCN5 protects β-cells, but only in male mice. This protection cannot be solely attributed to the pro-islet effect and suggests the involvement of extra-pancreatic and sex-specific roles. This study indirectly screened normal CCN5 gene expression by detecting β-galactosidase activity, measured in X-gal-stained tissues before fixation and sectioning. In order to create the gene deletion through homologous recombination, LacZ was inserted between exons 2 and 5 of the CCN5 gene. As a result, male knockout mice exhibited a distinct blue staining in the pseudostratified columnar epithelium with stereocilia of the epididymis and vas deferens. This type of epithelium comprises a single layer of cells but appears to have multiple layers due to the nuclei being at different levels. The epithelium possesses long projections made of actin filaments and plays a crucial role in absorbing, transporting, and maturing sperm cells. The staining was also clear in Leydig cells of the testis, which produce testosterone, which was confirmed using immunohistochemistry and authentic CCN5 antibody. In female mice, however, only a few cells in the ovary and scattered staining was observed in the endometrial glands of the uterus. Since androgens promote energy expenditure, lipolysis, and insulin sensitivity, knockout or overexpression of CCN5 may potentially affect androgen and/or sperm production, leading to metabolic consequences in males. Our results from in vitro, knockout, and overexpression models support the notion that CCN5 promotes β-cell growth and survival against diabetes and further suggest that it possesses sex-specific, metabolic effects. Presentation: 6/2/2024

12547 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024

12674 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024

6432 Enriching Testicular Organoid Aggregates to Promote Germ Cell Homing

Abstract Disclosure: A. Patel: None. O.O. Printy: None. C. Joswiak: None. M.M. Laronda: None. The long-term effects of life-saving chemotherapy treatments, such as an increased risk of infertility, are of concern to the growing number of childhood cancer survivors. De novo testicular morphogenesis seeks to generate testicular organoids that are structurally and functionally similar to the native testis. These organoids serve as a tool for investigating patient-specific models for restoring spermatogenesis for prepubertal cancer patients. Our lab has previously developed testicular organoids containing tubule-like structures (TLS) resembling the native seminiferous tubules as assessed by immunohistochemistry of testicular cell-type markers for Leydig cells (HSD3B2), peritubular myoid cells (ACTA1), Sertoli cells (SOX9), germ cells (DDX4), and spermatogonial stem cells (SSCs) (SALL4). Internal architecture demonstrated semi-continuous ring-like structures composed of ACTA1-positive cells encapsulating a population of SOX9-positive cells with HSD3B2-positive cells clustering near the periphery of the organoids. However, organoids lacked native germ cell populations. We hypothesized that murine testicular organoids would merge to form elongated TLS, within which exogenous SSCs would home to existing Sertoli cell niches, when cultured within structures designed from the appropriate dimensions and materials. Organoids were generated from testicular tissue isolated from 5 days-post-partum (dpp) CD1 mice using cell-seeding densities and microenvironmental conditions previously established by our lab. To promote aggregate formation, 48-hour-old organoids were transferred to trough-shaped structures formed from 2% agarose using 3D printed silicone molds. Separately, SSCs were isolated from 5 dpp tdTomato+ mice via magnetic-activated cell sorting of THY1+ cells. Organoids and aggregates were enriched with SSCs by three methods: (1) enrichment of cell suspension during organoid formation, (2) enrichment of culture during aggregate formation, and (3) injection of SSCs into aggregate using 20 µm glass pipette. Organoids transferred to agarose troughs on day 2 of culture merged to form aggregates by day 5 and continued to display dynamic architecture, shortening in length and increasing in width over time. Aggregates were more elongated in shape when cultured in narrow troughs less than 700µm in width. Observations thus far indicate that attempts to inject aggregates resulted in penetrance and inclusion of the SSC suspension; additionally, tdTomato+ cells were visualized within injected aggregates on day 17 of culture. Research is ongoing to define where exogenous SSCs take up residence, how long they survive, and if they proliferate over time within the testicular aggregates. This research expands upon foundational work establishing testicular organoids as an approach to in vitro spermatogenesis with clinical applications in fertility restoration. Presentation: 6/2/2024

7696 Ovarian Steroid Cell Tumor Presenting As Non-classical Adrenal Hyperplasia

Abstract Disclosure: V. Reddy: None. A. Gowda: None. V. Vattipally: None. D. Kunduru: None. Ovarian Steroid Cell Tumor Presenting as Non-classical Adrenal Hyperplasia IntroductionSteroid cell tumors(SCT) of the ovary make up less than 0.1% of all ovarian tumors. The three subtypes are stromal luteoma, Leydig cell tumor, and steroid cell tumor-not otherwise specified (NOS) which is the commonest subtype. Androgen excess is seen in 75% tumors and presents with virilization. Tumors are usually benign but maybe malignant in 25-43% cases. Factors predicting malignancy include size >7 cm, mitotic activity, necrosis, hemorrhage, and nuclear atypia. Case ReportA 29 y/o Caucasian female presented with secondary amenorrhea and hirsutism. History includes DM2,hypothyroidism,depression and morbid obesity. She reported early pubarche by age 8,menarche at age 13 and developed hirsutism and irregular menstrual cycles from age 15. No workup due to poor follow up until she saw PCP at age 21.She was diagnosed with PCOS and started on Metformin. Eventually referred to endocrinology at age 27, since amenorrhea and hirsutism persisted. On exam she had significant male pattern hair loss and hirsutism over face and body.She reported three first trimester miscarriages.Labs showed testosterone 370 ng/dl, 17 OHP 808 ng/dl, DHEAS 47 ng/dl, Estradiol 117 pg/ml, FSH 2.2 mIU/ml, LH 2.5 mIU/ml, androstenedione 319 ng/dl.Transvaginal US was unremarkable. She was diagnosed with non classical adrenal hyperplasia (NCAH) and started on treatment with prednisone 5 mg daily, drospirenone and spironolactone 100 mg daily.3 months later, labs showed 50% lowering of testosterone to 199 ng/dl and 17 OHP to 407 ng/dl.There was continued biochemical and clinical improvement at the next visit with 17 OHP 73 ng/dl and testosterone 55 ng/dl. Labs done 21 months after initial presentation, showed sudden dramatic rise in 17 OHP to 1148 ng/dl and testosterone to 639 ng/dl. Normal AFP, HCG, CEA, CA125, CA19-9, Inhibin A. CT showed 10.4 cm solid right ovarian mass and underwent right salpingo-oophorectomy. Pathology showed steroid cell tumor, NOS. Labs 1 month postoperatively showed complete normalization of testosterone to <3 ng/dl, 17 OHP to 11 ng/dl and DHEAS 0.66 ng/dl. Steroid therapy was tapered and discontinued. DiscussionWe present the case of a patient with ovarian SCT presenting as NCAH.SCTs are very rare accounting for only 0.1% of all ovarian tumors. Since they commonly secrete androgens, they present similarly to CAH or PCOS and may rarely even co-exist with CAH. Differentiating between NCAH and SCT is difficult especially when there is initial response to steroid therapy and imaging is unremarkable as with our patient. It is important to consider SCT in patients with hyperandrogenism even in the setting of elevated 17 OHP levels. Care should be taken to follow up both clinical and metabolic metrics closely and investigate if any change is noticed. Early diagnosis can prevent irreversible symptoms of hyperandrogenism. Presentation: 6/1/2024

12674 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024

6432 Enriching Testicular Organoid Aggregates to Promote Germ Cell Homing

Abstract Disclosure: A. Patel: None. O.O. Printy: None. C. Joswiak: None. M.M. Laronda: None. The long-term effects of life-saving chemotherapy treatments, such as an increased risk of infertility, are of concern to the growing number of childhood cancer survivors. De novo testicular morphogenesis seeks to generate testicular organoids that are structurally and functionally similar to the native testis. These organoids serve as a tool for investigating patient-specific models for restoring spermatogenesis for prepubertal cancer patients. Our lab has previously developed testicular organoids containing tubule-like structures (TLS) resembling the native seminiferous tubules as assessed by immunohistochemistry of testicular cell-type markers for Leydig cells (HSD3B2), peritubular myoid cells (ACTA1), Sertoli cells (SOX9), germ cells (DDX4), and spermatogonial stem cells (SSCs) (SALL4). Internal architecture demonstrated semi-continuous ring-like structures composed of ACTA1-positive cells encapsulating a population of SOX9-positive cells with HSD3B2-positive cells clustering near the periphery of the organoids. However, organoids lacked native germ cell populations. We hypothesized that murine testicular organoids would merge to form elongated TLS, within which exogenous SSCs would home to existing Sertoli cell niches, when cultured within structures designed from the appropriate dimensions and materials. Organoids were generated from testicular tissue isolated from 5 days-post-partum (dpp) CD1 mice using cell-seeding densities and microenvironmental conditions previously established by our lab. To promote aggregate formation, 48-hour-old organoids were transferred to trough-shaped structures formed from 2% agarose using 3D printed silicone molds. Separately, SSCs were isolated from 5 dpp tdTomato+ mice via magnetic-activated cell sorting of THY1+ cells. Organoids and aggregates were enriched with SSCs by three methods: (1) enrichment of cell suspension during organoid formation, (2) enrichment of culture during aggregate formation, and (3) injection of SSCs into aggregate using 20 µm glass pipette. Organoids transferred to agarose troughs on day 2 of culture merged to form aggregates by day 5 and continued to display dynamic architecture, shortening in length and increasing in width over time. Aggregates were more elongated in shape when cultured in narrow troughs less than 700µm in width. Observations thus far indicate that attempts to inject aggregates resulted in penetrance and inclusion of the SSC suspension; additionally, tdTomato+ cells were visualized within injected aggregates on day 17 of culture. Research is ongoing to define where exogenous SSCs take up residence, how long they survive, and if they proliferate over time within the testicular aggregates. This research expands upon foundational work establishing testicular organoids as an approach to in vitro spermatogenesis with clinical applications in fertility restoration. Presentation: 6/2/2024

5116 Postmenopausal Hyperandrogenism due to Rare Ovarian Tumor

Abstract Disclosure: M. Salim: None. S. Dasaraju: None. Y. Lee: None. S. Afzal: None. B.K. Erickson: None. M.A. Khalifa: None. L.A. Burmeister: None. Background: Hyperandrogenism in postmenopausal women may arise from either ovarian or adrenal source and can pose challenging diagnostic dilemma. Clinical Case: A 66-year-old multigravida, post-menopausal woman presented with a 3-month history of worsening alopecia. Her past medical history included diabetes mellitus type 2 and breast cancer. Menarche was at age 12. There was no history of infertility. Menopause was in her 40’s. She had been shaving her upper lip and chin hair every 3 days for the past 10 years. Scalp hair loss had accelerated in the last 3 months, with loss of the bangs. Physical exam was significant for body mass index 34, deep voice, and clitoromegaly. Laboratory tests showed testosterone 160 ng/dL, (8-60 ng/dL), free testosterone 3.37 ng/dL (0.06-0.38 ng/dL), androstenedione 2.196 ng/mL (0.13-0.82 ng/mL), sex hormone binding globulin (SHBG) 31 nmol/L (30-135 nmol/L), dehydroepiandrosterone-sulfate (DHEAS) 86 ug/dL (13-130 ug/dL), 17-hydroxy progesterone 96 ng/dL (<51 ng/dL), inhibin A 1.1 pg/mL (<6.9 pg/mL), inhibin B <10 pg/mL (<16 pg/mL), adrenocorticotropic hormone 38 pg/mL (<47 pg/mL), and cortisol 12.2 ug/dL (4-22 ug/dL). Response to 1 mg overnight dexamethasone suppression test demonstrated persistently elevated testosterone (160 ng/dL), and incomplete suppression of androstenedione (0.827 ng/mL) and cortisol (2.4 ug/dL). Computerized tomography (CT) scan showed a left adrenal nodule (12 mm, 5 Hounsfield units, stable compared to imaging 2 years prior) and unremarkable appearance of the ovaries. Pelvic ultrasound did not show ovarian tumor on the right and left ovary was not seen. Adrenal and ovarian vein sampling suggested the ovaries as the source of the testosterone. Given the ovarian vein sampling results, a multidisciplinary discussion between endocrinology and gynecologic oncology concluded that bilateral salpingo-oophorectomy (BSO) was the next best step for diagnosis and management. Laparoscopic BSO was performed. Intraoperatively, the ovaries appeared grossly normal with the exception of dilated ovarian veins. Histopathology showed bilateral Leydig cell tumors (left: 1.5 cm; right: 2.0 cm) and a 0.7 cm left ovarian Brenner tumor. At 1-year postoperative follow-up, alopecia had resolved. Laboratory evaluation showed normalization of the testosterone (23 ng/dL), free testosterone (0.47 ng/dL). Androstenedione (1.023 ng/mL) was lower than before surgery but higher than reference range. Conclusion: The presented rare case of postmenopausal virilization is even more unique due to the synchronous presence of 2 rare ovarian tumors, both capable of causing hyperandrogenism, including bilateral ovarian Leydig cell tumor, Brenner tumor and adrenal incidentaloma. Despite normal appearing ovaries on imaging studies, BSO was ultimately necessary for diagnosis and management. Presentation: 6/3/2024

6450 Leydig Cell Tumor: A Rare Cause of Post-menopausal Hyperandrogenism

Abstract Disclosure: R. Nakdali: None. S. Athimulam: None. Introduction: Ovarian steroid cell tumors are a rare subtype of sex-cord stromal tumors. Leydig cell tumors is a subtype of sex-stromal tumors that is found in < 1% of all ovarian tumors. They are typically benign, unilateral, and secrete androgens which causes virilization. Below we present the case of a postmenopausal woman who presented with signs of virilization and was diagnosed with an ovarian Leydig cell tumor. Case: A 62-year-old postmenopausal woman, with a history of partial hysterectomy with right oophorectomy presented to Endocrinology clinic for evaluation of thyroid nodules. However, clinically she had signs of virilization noted by the provider on examination. She reported progressive androgenic alopecia, cystic acne, oily skin, hoarseness of voice and hirsutism, with increase hair growth over upper lip, chin and jaw line requiring daily shaving. She denied taking any supplements. She reported embarrassment of these physical changes and prior providers had attributed this to aging, therefore not prompting further testing. This caused significant emotional distress to the patient, leading to isolation. Biochemical testing confirmed elevated bioavailable testosterone (193.1 ng/dL) and total testosterone levels (579 ng/dL), with no evidence of hypercortisolemia. Magnetic resonance imaging (MRI) revealed a heterogenous soft tissue mass (2.9 x 2.1 x 2.1 cm) in the left ovary. She underwent a left salpingo-oophorectomy and pathology confirmed a well-differentiated 2.4 cm Leydig cell tumor. Post-operatively, she reported improvement in facial and body hirsutism, reduced shaving frequency decrease in androgenic hair loss, with noticeable hair growth in her frontal scalp, and her skin became less oily with reduced acne. Post-operative labs confirmed cure of hyperandrogenism (Bioavailable testosterone < 2.6 ng/dL; Total testosterone: < 10ng/dL; and Free androgen index < 0.5%). She has been referred to genetics for evaluation. Discussion: Post-menopausal hyperandrogenism poses a diagnostic challenge as it can be mistaken for hormonal imbalances seen with aging. Often times, patients do not report symptoms due to embarrassment unless specifically addressed by provider. Initial testing consists of total and free testosterone and dehydroepiandrosterone sulfate (DHEAS) levels to assess the source of hyperandrogenism. Ruling out hypercortisolemia and assessment for ingestion of testosterone or DHEA supplements is vital. Pelvic ultrasound or cross-sectional imaging (CT or MRI) of abdomen and pelvis can identify structural abnormalities in the ovaries or adrenal glands. Prompt evaluation and management can alleviate significant distress to the patient. Leydig cell tumors represent a rare but important consideration in the differential diagnosis of postmenopausal hyperandrogenism, such as in our patient. Presentation: 6/2/2024

12465 Unraveling The Enigma: A Multifaceted Presentation Of Hormonal Dysregulation In A 37-year-old Woman

Abstract Disclosure: S.Y. Rizvi: None. N. Ahmad: None. Title: Unraveling the Enigma: A Multifaceted Presentation of Hormonal Dysregulation in a 37-Year-Old Woman Background: Leydig cell tumors (LCTs) are rare gonadal tumors, arising from the interstitial cells of the testis or, less commonly, the ovary, 0.1%. These tumors are known for their potential to produce androgens, leading to virilization symptoms such as hirsutism. While LCTs are typically benign, they can occasionally exhibit malignant features. Accurate diagnosis and appropriate management are crucial to prevent complications and ensure favorable outcomes. Clinical Case: A 37-year-old, G3P3A0 woman, presented to our endocrinology clinic for a follow-up visit following the removal of a malignant thyroid nodule. During the visit, her endocrinologist noted elevated testosterone levels and worsening hirsutism. She reported mild abdominal pressure but denied other symptoms. She also complained of pain in the lower abdomen, although physical examination findings were limited due to type II obesity. Diagnostic investigations revealed a history of imaging studies, including CT scans and pelvic ultrasounds, demonstrating various findings such as a fibroid uterus, ovarian abnormalities, and a heterogeneous mass in the left adnexa. Laboratory evaluations over time showed fluctuations in androgen levels, with notable values including DHEA-sulfate (137.5 ug/dL), total testosterone (309.6 NG/dL), and free testosterone (12.85 NG/dL). Tumor markers, including CA 19-9= 16 U/mL, CA 125= 23.3 U/mL, CEA = 1.7 NG/mL, and HCG, were unremarkable. Gynae/Oncology was consulted, and she underwent surgical exploration with laparoscopic left oophorectomy. It revealed a Leydig cell tumor without overt malignant features, as confirmed by histopathological examination and immunohistochemical analysis. Her testosterone improved since oophorectomy. Patient undergoes frequent post-op follow up of her testosterone, free+total lc/MS to monitor for hyperandrogenism. Conclusion: This case highlights the importance of considering Leydig cell tumors in the differential diagnosis of hirsutism, particularly in the presence of elevated androgen levels. Multidisciplinary collaboration involving endocrinologists, gynecologists, oncologists, radiologists, and pathologists is essential for accurate diagnosis and optimal management. Surgical resection remains the cornerstone of treatment for Leydig cell tumors, with careful histopathological evaluation necessary to assess for potential malignancy. Long-term follow-up is warranted to monitor for recurrence or metastasis, although the prognosis for benign Leydig cell tumors is generally favorable. Patients with large body habitus may present challenges in physical examination, necessitating reliance on imaging and laboratory studies for diagnostic evaluation. Keywords: hirsutism, testosterone, ovary Presentation: 6/2/2024

8057 Unveiling Gender Dysphoria and Gender Change in a Large Cohort of DSD

Abstract Disclosure: R.L. Batista: None. M. Inacio: None. T.A. Bachega: None. N.L. Gomes: None. G. Madureira: None. M.C. Miranda: None. R.T. dallago: None. M.M. Ferrari: None. L.M. Lousada: None. J.A. Batatinha: None. E.F. Costa: None. M.P. Sircili: None. F. Denes: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. Disorders/Differences of Sex Development (DSD) encompass a wide range of genetic conditions that affect sex development. Individuals with DSD may experience gender dysphoria and undergo gender changes throughout their lives. To analyze gender dysphoria and gender changes in individuals with DSD, we conducted a retrospective study involving all DSD cases from a single Brazilian tertiary medical center followed by a multidisciplinary team. We included 696 subjects considering their DSD diagnosis, sex assignment, age at DSD diagnosis, gender change, age at gender change, and phenotypic features. Among the three categories of DSD, chromosomal DSD was diagnosed in 264 subjects (135 with a 45,X karyotype, 101 mosaics with 45,X/46,Xi(Xq)), and three exhibited chimerism (all with ovotesticular DSD). Thirteen patients with chromosomal DSD and Y material were assigned as male, while two patients initially assigned as female underwent gender changes. Among the 258 subjects with 46,XY DSD, 69 had undetermined DSD, 64 had gonadal dysgenesis, 36 had 5-αRD2 deficiency, 18 had 17β-HSD3 deficiency, 11 had 17α-hydroxylase deficiency, 9 had Leydig cell hypoplasia, 25 had CAIS, 18 had PAIS, and 8 had AMH defects. Out of the 192 XY subjects with atypical genitalia, 89 (46%) were raised as female, and 13% changed from female to male, predominantly in cases of 5αRD2 deficiency (45%), followed by 17β-HSD3 deficiency (33%). Only 2.9% of those raised as male changed to female gender. Among the 46,XX DSD cases, congenital adrenal hyperplasia (CAH), mainly 21-hydroxylase deficiency, was diagnosed in 123 cases (115 initially assigned as female). Six cases of CAH resulted in gender changes from female to male, notably in the virilizing salt-wasting (VS) form (5/6; p=.004), all of which had a late onset of treatment (>2 years old) and poor compliance. Among the remaining 55 cases with 46,XX DSD, 24 had ovotesticular DSD (all with atypical genitalia), while 16 had 46,XX testicular DSD (seven with atypical genitalia); four cases underwent gender changes, with three changing from female to male. Twelve patients with 46,XX DSD had an undetermined diagnosis (all with atypical genitalia, three with syndromic features). Additionally, one patient had aromatase deficiency, one had POR deficiency, and one had glucocorticoid resistance syndrome. In conclusion, our study highlights a clear trend: gender changes, notably from female to male, were more prevalent in 46,XY DSD, particularly in 5αRD2 and 17β-HSD3 deficiencies. This underscores the importance of considering male sex assignment for individuals with these diagnoses. Additionally, factors such as the VS form of CAH, delayed treatment initiation, and inadequate compliance significantly contributed to female-to-male gender changes among 46,XX DSD cases. These findings offer critical insights guiding more effective and targeted interventions for DSD individuals. Presentation: 6/2/2024

9243 Postmenopausal Hyperandrogenism due to Rare Ovarian Tumor

Abstract Disclosure: M. Salim: None. S. Dasaraju: None. Y. Lee: None. S. Afzal: None. B.K. Erickson: None. M.A. Khalifa: None. L.A. Burmeister: None. Background: Hyperandrogenism in postmenopausal women may arise from either ovarian or adrenal source and can pose challenging diagnostic dilemma. Clinical Case: A 66-year-old multigravida, post-menopausal woman presented with a 3-month history of worsening alopecia. Her past medical history included diabetes mellitus type 2 and breast cancer. Menarche was at age 12. There was no history of infertility. Menopause was in her 40’s. She had been shaving her upper lip and chin hair every 3 days for the past 10 years. Scalp hair loss had accelerated in the last 3 months, with loss of the bangs. Physical exam was significant for body mass index 34, deep voice, and clitoromegaly. Laboratory tests showed testosterone 160 ng/dL, (8-60 ng/dL), free testosterone 3.37 ng/dL (0.06-0.38 ng/dL), androstenedione 2.196 ng/mL (0.13-0.82 ng/mL), sex hormone binding globulin (SHBG) 31 nmol/L (30-135 nmol/L), dehydroepiandrosterone-sulfate (DHEAS) 86 ug/dL (13-130 ug/dL), 17-hydroxy progesterone 96 ng/dL (<51 ng/dL), inhibin A 1.1 pg/mL (<6.9 pg/mL), inhibin B <10 pg/mL (<16 pg/mL), adrenocorticotropic hormone 38 pg/mL (<47 pg/mL), and cortisol 12.2 ug/dL (4-22 ug/dL). Response to 1 mg overnight dexamethasone suppression test demonstrated persistently elevated testosterone (160 ng/dL), and incomplete suppression of androstenedione (0.827 ng/mL) and cortisol (2.4 ug/dL). Computerized tomography (CT) scan showed a left adrenal nodule (12 mm, 5 Hounsfield units, stable compared to imaging 2 years prior) and unremarkable appearance of the ovaries. Pelvic ultrasound did not show ovarian tumor on the right and left ovary was not seen. Adrenal and ovarian vein sampling suggested the ovaries as the source of the testosterone. Given the ovarian vein sampling results, a multidisciplinary discussion between endocrinology and gynecologic oncology concluded that bilateral salpingo-oophorectomy (BSO) was the next best step for diagnosis and management. Laparoscopic BSO was performed. Intraoperatively, the ovaries appeared grossly normal with the exception of dilated ovarian veins. Histopathology showed bilateral Leydig cell tumors (left: 1.5 cm; right: 2.0 cm) and a 0.7 cm left ovarian Brenner tumor. At 1-year postoperative follow-up, alopecia had resolved. Laboratory evaluation showed normalization of the testosterone (23 ng/dL), free testosterone (0.47 ng/dL). Androstenedione (1.023 ng/mL) was lower than before surgery but higher than reference range. Conclusion: The presented rare case of postmenopausal virilization is even more unique due to the synchronous presence of 2 rare ovarian tumors, both capable of causing hyperandrogenism, including bilateral ovarian Leydig cell tumor, Brenner tumor and adrenal incidentaloma. Despite normal appearing ovaries on imaging studies, BSO was ultimately necessary for diagnosis and management. Presentation: 6/3/2024

Testosterone and 5 Alpha Reductase Inhibitor (5ARI) in Benign Prostatic Hyperplasia (BPH): A historical perspective

This paper provides a review of the historical and scientific evolution of testosterone and 5ARI research. It chronicles the evolution from early empirical practices like castration, through the formal discovery of testosterone in the 20th century, to the eventual synthesis of testosterone and identification of Leydig cells. Testosterone’s crucial role in male development and its influence on the development and maturation process of the prostate gland are also presented. The introduction of 5ARIs in the 1990s, such as finasteride, marked a significant advancement in managing BPH by reducing dihydrotestosterone (DHT) levels. The controversy regarding the therapeutic use of 5ARIs is discussed, given concerns about their association with high-grade prostate cancer and other systemic risks. Likewise, emerging evidence challenges the traditional view that testosterone exacerbates prostate conditions, suggesting that testosterone replacement therapy (TRT) may improve symptoms of low testosterone without increasing BPH symptoms, prostate cancer (PCa), or cardiovascular risk. Several new studies are discussed suggesting that low or declining testosterone level could be a risk factor for prostate tumorigenesis. This review emphasizes the need for continued research on Testosterone and 5ARI to further define their role in men’s health.

Taste receptor T1R3 regulates testosterone synthesis via the cAMP-PKA-SP1 pathway in testicular Leydig cells

Taste receptor type 1 subunit 3 (T1R3) is a G protein-coupled receptor encoded by the TAS1R3 gene that can be specifically activated by certain sweeteners or umami agents for sweet/umami recognition. T1R3 is a potential target for regulating male reproduction. However, studies on the impact of non-nutritive sweeteners on reproduction are limited. In the present study, we evaluated the impact of the non-nutritive sweeteners (saccharin sodium, sucralose and acesulfame-K) on testosterone synthesis in testicular Leydig cells of Xiang pigs by comparing the relative abundance of mRNA transcripts and protein expression of T1R3, steroidogenic related factors, and intracellular cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), as well as testosterone levels using Western blotting, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). To clarify the specific mechanism, a dual luciferase assay was used to uncover the relationship between the transcription factors and steroidogenic enzyme. The acute intratesticular injection of a typical non-nutritive sweeteners was conducted to verify this impact in mouse. The results showed that saccharin sodium not only enhanced T1R3 expression in Leydig cells of Xiang pigs, but also caused significant increases in testosterone, cAMP, PKA, phosphorylation of specificity protein 1 (p-SP1), total protein of specificity protein 1 (SP1), steroidogenic acute regulatory protein (StAR), and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) (P<0.05). Similarly, treatment of Leydig cells with sucralose and acesulfame-K also increased testosterone level, protein expression of T1R3, 17-α-hydroxylase/17, 20-lyase (CYP17A1), and 3β-HSD1 (P<0.05). Treatment with SQ22536 (an adenylate cyclas inhibitor) or H89 (a PKA inhibitor) significantly reduced saccharin sodium-induced protein levels of p-SP1, StAR, CYP17A1, and 3β-HSD1 (P<0.05). In addition, a dual luciferase assay further demonstrated that SP1 significantly increased the promoter activity of CYP17A1 (P<0.05). When mouse testes were injected with saccharin sodium, T1R3, p-SP1, CYP17A1, and 3β-HSD1 were upregulated, leading to a significant testicular increase in testosterone and cAMP levels (P<0.05). These results suggest a mechanism by which the taste receptor T1R3 regulates testosterone production, and this mechanism may be linked to the cAMP-PKA pathway. Understanding the interrelationship between T1R3 and the cAMP-PKA-SP1 pathway contributes to clarify the regulatory mechanisms of male reproduction.

Deficiency in the Rab25 gene leads to a decline in male fertility and testicular injury: Impact on the regulation of germ cell proliferation and apoptosis

BackgroundRab25 is a member of the Rab family, functioning as a regulatory molecule in intracellular transport. Although its involvement in cellular functions and disease development is well-established, its precise roles in male reproductive physiology remain elusive. MethodsTo explore the specific roles of Rab25 in testicular development and spermatogenesis, we established the Rab25−/− mouse model and Rab25 knockdown germ cell line (GC-2). We compared the fertility, sperm analysis, and testicular tissues between Rab25−/− and wild-type male mice. To delve deeper into potential mechanisms, we employed immunohistochemistry, TUNEL assay, Western Blotting, CCK-8 assay, etc. to evaluate cell proliferation and apoptosis in testicular tissues and GC-2 cells. ResultsOur findings indicated that Rab25 was expressed in germ cells and Leydig cells in the testes. Although the weight of Rab25−/− mice testes exhibited no significant changes, fertility was compromised, with a decrease in sperm quantity and reduced motility. HE staining revealed a disorganized arrangement of germ cells and vacuolization. Additionally, chromatin marginalization and nuclear pyknosis were observed in the Rab25−/− mice. In both Rab25−/− mice testes and Rab25 knockdown GC-2 cells, we found that germ cell proliferation was reduced, while apoptosis was increased. ConclusionsIn conclusion, our study proposes that Rab25 plays a vital role in spermatogenesis by regulating the proliferation and apoptosis of germ cells.

The role of Sertoli cell-derived miR-143-3p in male fertility declines with age

As delayed parenthood becomes more prevalent, understanding age-related testosterone decline and its impact on male fertility has gained importance. However, molecular mechanisms concerning testicular aging remain largely undiscovered. Our study highlights that miR-143-3p, present in aging Sertoli cells (SCs), is loaded into extracellular vesicles (EVs), affecting Leydig cells (LCs) and germ cells, thus disrupting testicular tissue homeostasis and spermatogenesis. Intriguingly, in SCs, TGF-β signaling promotes miR-143 precursors transcription, increasing mature miR-143-3p levels. This inhibits Smurf2, activating Smad2, and further enhancing miR-143-3p accumulation. EVs transporting miR-143-3p, originating from SCs, contribute to the age-related decline of testosterone and male fertility by targeting the luteinizing hormone receptor and retinoic acid receptor. Diminishing endogenous miR-143-3p in SCs postpones testis aging, preserving and prolonging male fertility. Thus, our study identified miR-143-3p as a key regulator of testicular function and fertility, revealing miR-143-3p as a potential therapeutic target for male abnormal sexual and reproductive function.

HSF1/HSP25 system protects mitochondria function from heat stress and assists steroidogenesis in MA-10 Leydig cells

Heat shock response is characterized by the induction of heat shock proteins (HSPs) or molecular chaperones that maintain protein homeostasis. Heat shock transcription factor 1 (HSF1) plays a central role in heat shock response in mammalian cells. To investigate the impact of the heat shock response mechanism on steroidogenesis, we generated MA-10 mouse Leydig tumor cells deficient in HSF1 using CRISPR-Cas9 genome editing. Under heat stress conditions, the levels of StAR protein, but not its mRNA, decreased more in HSF1-knockout cells than in wild-type cells, confirming that HSF1 stabilizes StAR protein. Simultaneously, HSP110, HSP70, and HSP25 were markedly upregulated in an manner dependent on HSF1. Mitochondrial membrane potential (MMP) and ATP synthesis were decreased in HSF1-knockout cells under heat stress conditions, and mitochondrial fragmentation was enhanced. Furthermore, treatment with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a disruptor of MMP, reduced the levels of StAR protein to a greater extent in HSF1-knockout cells than in wild-type cells, which was associated with decreased MMP and ATP synthesis. Unexpectedly, HSP25 expression was markedly increased in wild-type cells following CCCP treatment. HSP25 knockdown reduces MMP under heat stress conditions and decreases StAR protein levels and progesterone synthesis. HSP25 overexpression in HSF1KO cells restored StAR protein levels. These results show that the HSF1/HSP25 pathway protects mitochondrial function and maintains StAR synthesis.

ATF3 as a response factor to regulate Cd-induced reproductive damage by activating the NRF2/HO-1 ferroptosis pathway

Cadmium (Cd) has garnered significant attention due to reproductive toxicity in inducing ferroptosis. However, the specific mechanisms underlying Cd-induced germ cell ferroptosis remain poorly understood. This study aimed to systematically explore the molecular mechanisms of germ cell ferroptosis by investigating differential changes in transcription factors and proteins in male mice treated orally with CdCl2 (0.5 g/L) reaching postnatal day 60, alongside Leydig cell (TM3) and Sertoli cell (TM4) lines. Results demonstrated that Cd exposure led to increased iron overload and oxidative stress in mouse testes, disrupted intracellular mitochondrial morphology characteristic of ferroptosis. RNA sequencing revealed significant upregulation of Atf3 and Hmox1 in Cd-exposed germ cells, along with increased expression of ATF3 and HO-1. Intervention in ferroptosis or HO-1 effectively rescued cells from Cd-induced mortality by breaking the detrimental cycle between lipid peroxidation and HO-1 activation. Further findings showed that NRF2 and HO-1 expression was notably elevated upon ATF3 overexpression in TM3 and TM4 cells, activating the Keap1-Nrf2 pathway and triggering ferroptosis in testes, whereas NRF2 and HO-1 expression levels were reversed when ATF3 was silenced. This study provides novel insights into ATF3-mediated NRF2/HO-1 signaling in Cd-induced mitochondrial ferroptosis in testes, shedding light on the mechanisms underlying Cd-induced ferroptosis and testicular injury.

Therapeutic Effect of Nano-Extract of Nigella Sativa Seeds on The Histological Structure of The Testis In Male Albino Rats Induced With Anemia

Background: The study explores the effect of nanoparticles derived from the aqueous extract of Nigella sativa seeds on the histological structure of the testis in male albino rats with tartrazine-induced anemia. Nanoparticles, ranging in size from 1 to 100 nanometers, possess unique physical and chemical properties that enhance their ability to interact and deliver drugs. Objective: This study focuses on the therapeutic potential of Nigella sativa seed extract nanoparticles in treating testicular histological changes in anemic rats. The testicle is an oval organ involved in sperm formation and hormone production. It contains seminiferous tubules, Sertoli cells, and Leydig cells, which are responsible for sperm production and testosterone synthesis. Methods: The study included 36 male albino mice, aged between 8-12 weeks, and weighing 240-260 grams, from the Iraqi Center for Cancer Research and Medical Genetics. The animals were divided into six groups, each group having six animals. Results: The study showed that tartrazine at a concentration of 20 mg/kg: with Nigella sativa nanoparticles 50 mg/kg: an increase in weight. the body. While tartrazine at a concentration of 20 mg/kg with N.sativa nanoparticles at 75 mg/kg did not show significant differences compared with the control group. The results showed the Nigella sativa seed nanoparticles at a concentration of (50 mg/kg, 75 mg/kg): showed an increase in body weight, while tartrazine at a concentration of 20 mg/kg: a decrease in body weight, and no significant changes were observed in testicle weight in all treatments compared with the control group. Non-significant changes in testicle weight compare with Control group. The study showed that treatment with tartrazine 20 mg/kg showed noticeable histological changes in the histological structure of the testicle. Tartrazine with N.sativa nanoparticles (50 mg/kg, 75 mg/kg): No histological changes, indicating attenuation of tartrazine-induced damage. N.sativa nanoparticles (50 mg/kg, 75 mg/kg): demonstrated normal composition of testicular tissue, indicating no harmful effects on testicular tissue. has protective effects. Conclusion:The study aimed to investigate the potential of using nanoparticles extracted from Nigella sativa seeds to protect against damage caused by tartrazine in male albino rats.with significant therapeutic implications for the use of natural antioxidants in biomedical applications.

Histological Study of Development Structure of Testis and Epididymis in Gazelle Gazelle Subgutturosa

Objective: The aim of this study for focused the light on the development structure of the testis of gazelle. Materials and Method: 10 healthy male gazelle were used to study the histology structure of testis (5 pre- Puberty and 5 post- Puberty). Results: The testis is surrounded by a thick, irregular connective tissue capsule. Each lobule of the testis is composed of seminiferous tubules and interstitial tissue, and trabecular and interlobular septa divide the lobules apart. Neighboring the seminiferous tubules are Sertoli cells and spermatogenic cells. From spermatogenic cells, spermatozoa are produced. The first spermatogonia are small, spherical cells with black, spherical nuclei that reside on the basement membrane. Spermatogonia were earliest stages of the spermatogenic cells. Larger cells with frequently distinct chromatin are called primary spermatocytes. Secondary spermatocytes are rare because of their quick second meiotic division and the haploid spermatids that follow. Spermatids are round, pale-nucleated cells that are grouped close to the lumen of the seminiferous tubule. Prostatogenic cells outnumber Sertoli cells in the body. Oval or triangular in shape, the nucleus has a light coloration. In the connective tissue separating adjacent tubules, Leydig cells are present. Lines the epididymis are pseudostratified epithelium. Extensions of the cytoplasm into the lumen are called stereocilia. In different proportions, there is circular smooth muscle, and a connective tissue lamina propria supports the epithelium. The rete testis is connected to the ductus epididymidis by the efferent ductules, which are convolutions of tubules. Numerous valved veins were visible in the tunica albuginea, which covered the initial ascending portion of the epididymal head. Cuboidal epithelium was lining extratesticular rete testis. Three different cell types; migrating, basal, and columnar had epithelial membranes lining the efferent ductules. Many columnar, non-ciliated cells with many cytoplasmic vacuoles and tiny granules lined the epithelium of the first segment of the efferent ductules. Conclusion: In post-puberty age, the structure of the gazelle's testis exhibited a pattern resembling that of other ruminants; the seminiferous tubules contain adult sperm and a broad lumen.