Steroidogenic Pathway Research Articles

Prolactin locally mediates follicular atresia in hyperprolactinemic vizcachas (Rodentia, Chinchillidae)

Infertility in hyperprolactinemic females is attributed to the dysregulation of GnRH release, subsequently affecting gonadotropin levels, and ultimately leading to anovulation. However, in addition to the hypothalamus, prolactin receptor (PRLR) is expressed in ovaries as well, suggesting potential local effects of PRL in cases of hyperprolactinemia. We have developed an experimental model of sulpiride (SPD)-induced hyperprolactinemia using a wild rodent, the plains vizcacha, and studied the implications of pharmacological PRL levels on folliculogenesis and steroid production. Ovaries of SPD females showed a striking number of atretic follicles along with a reduction in the collective number of viable follicles leading to a higher atretic/viable follicle ratio compared to that of control females (CTL) (P < 0.05). In terms of sensitivity to the hormonal environment, SPD ovaries substantially changed their potential responsiveness to pituitary PRL, as evidenced by the three-fold increase in PRLR expression alongside heightened expression of both gonadotropic receptors in comparison to CTL ovaries (P < 0.05). Circulating estradiol (E2) values doubled post-treatment in the SPD females, which also showed higher expressions of aromatase and 17β-hydroxysteroid dehydrogenase, along with both E2 receptors, ERα and ERβ than the CTL group (P < 0.05). Our findings strongly suggest that hyperprolactinemia-dependent dysregulation of ovarian function can be explained at least partially, by PRL direct actions facilitated by the heightened expression of PRLR in follicles and corpora lutea. Possibly these PRL actions synergize with those triggered by gonadotropic hormones ultimately leading to alteration of the steroidogenic pathway, folliculogenesis disruption and increased atresia.

Methanolic extract of wheatgrass (Triticum aestivum L.) prevents BPA-induced disruptions in the ovarian steroidogenic pathway and alleviates uterine inflammation in Wistar rats.

The online version contains supplementary material available at 10.1007/s13205-024-04117-0.

New insights into the mechanisms underlying 5-hydroxymethylfurfural-induced suppression of testosterone biosynthesis in vivo and in vitro

5-hydroxymethylfurfural (HMF), produced by the Maillard reaction, can indicate thermal processes in food. Previous research has examined the cytotoxic, genotoxic, mutagenic, and carcinogenic characteristics of HMF and its derivatives in different organs. Nevertheless, there is currently no available evidence about the impact of HMF on male reproductive toxicity. In this study, the effects of HMF on testosterone biosynthesis in both mouse testis and TM3 Leydig cells were investigated. HMF was administered to mice at doses of 30 and 300 mg/kg/day for 21 days and to Leydig cells at concentrations of 0.1, 1, and 10 mM for 24 h. The mechanism of action of HMF on testosterone biosynthesis in both mouse testis and Leydig cells was revealed by measuring the amount of testosterone, 3′,5′-cyclic adenosine monophosphate (cAMP) levels, and the expression level of some important genes in the steroidogenic pathway. In addition, its effects on general testis were examined through histopathological evaluations. Upon examination of the results, it was observed that HMF had a significant impact on reducing testosterone and cAMP levels. Furthermore, HMF inhibited the expression of steroidogenic genes, including steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, 3β-hydroxy dehydrogenase, and 17β-hydroxy dehydrogenase, as well as transcription factors, such as steroidogenic factor-1, GATA binding protein-4, and nerve growth factor IB. HMF-administrated groups had germinal epithelium degradation, vacuolization, and disorders in the interstitial area. Consequently, it has been proven for the first time that HMF can damage the male reproductive system by detrimentally impacting the production of testosterone.

The impact of furan exposure on steroidogenesis in Leydig cells: cellular and molecular observations.

Furan is an organic compound that occurs as a result of heat treatment during the processing and cooking of many food products. Furthermore, the environment contains furan in tobacco smoke and vehicle exhaust gases, and it serves as an intermediate molecule in the synthesis of various pharmaceutical and chemical agents, pesticides, and stabilizers. Studies on the male reproductive system have not been able to elucidate the pathway through which furan exerts its negative effects. In this study, the TM3 Leydig cell line was exposed to various furan concentrations (0.03, 0.3, and 3 mM) for 24h. In order to assess the cytotoxic effects of furan on Leydig cells, we examined cell viability, cell proliferation, and lactate dehydrogenase enzyme levels. To investigate the detrimental effects of furan on testosterone biosynthesis, quantitative analyses were conducted on cAMP and testosterone levels, as well as the expression levels of key genes and transcription factors implicated in the steroidogenic pathway. The results indicate that furan inhibited the viability and proliferation of Leydig cells and enhanced the activity of lactate dehydrogenase. Leydig cells administered to furan exhibited notable reductions in cAMP and testosterone levels. Additionally, while the expression levels of steroidogenic genes were downregulated, significant changes were detected in the expression levels of the transcription factors responsible for the regulation of these genes. Consequently, our findings suggest that furan exerts inhibitory effects on steroidogenesis in Leydig cells through multiple mechanisms, ultimately leading to infertility by inducing dysfunction in Leydig cells.

7886 Impact of Withania somnifera (Ashwagandha) Supplement on Androgen Signalling Pathways

Abstract Disclosure: I.S. Barata: None. J. Yakubu: None. T. Du Toit: None. A.V. Pandey: None. Supplements derived from plant extracts are often used as alternatives to pharmaceutical drugs. These products are often perceived as safe despite the absence of scientific evidence regarding their efficacy and safety and are less strictly regulated than pharmaceutical products. Winter cherry (Withania somnifera, commonly known as ashwagandha) is used as a testosterone booster supplement in multiple countries. Withania somnifera extract was found to increase testosterone and androgen precursors in a small clinical study. However, there is a lack of data regarding effects of Withania somnifera on the steroidogenic pathways. Steroidogenesis impacts not only sexual differentiation, reproduction, and fertility but also other physiological processes, including hypertension and obesity. Moreover, the modulation of hormone signaling is an important target in hormone-responsive pathogenesis, and overproduction of androgens has been implicated in the pathogenesis of prostate cancer (PCa) and polycystic ovarian syndrome (PCOS). In humans, testosterone is mainly produced in the testes after conversion of pregnenolone to 17-hydroxypregnenolone (17-OHPreg) and dehydroepiandrosterone (DHEA, the precursor of androgens) by adrenal CYP17A1, which catalyzes both the 17-hydroxylation of steroids for cortisol production and the breakage of 17,20 bonds (17,20 lyase) in 17-OHPreg to produce DHEA. The intake of substances that may increase androgen levels by people with PCa or PCOS or with predisposition to these conditions may trigger pathogenesis or worsen prognosis by accelerating disease progression. We investigated the effects of Withania somnifera in modulating the adrenal steroidogenic pathway and the possible consequences on the pathogenesis of androgen-responsive conditions, specifically PCa. Adrenal cells were treated with the extract of Withania somnifera to characterize its effect on adrenal steroid profile and CYP17A1 hydroxylase and lyase activities. Prostate cancer proliferation was assessed by direct incubation of prostate cancer cells with extracts as well as with conditioned media from adrenal cells treated for 48h, to simulate natural steroidogenesis. The results suggest that adrenal steroid hormone biosynthesis can be altered by the intake of Withania somnifera extract and that this supplement may affect proliferation of PCa cells. Moreover, the reported testosterone booster effect of Withania somnifera supplementation does not appear to be through increased adrenal androgen production. Presentation: 6/1/2024

7886 Impact of Withania somnifera (Ashwagandha) Supplement on Androgen Signalling Pathways

Abstract Disclosure: I.S. Barata: None. J. Yakubu: None. T. Du Toit: None. A.V. Pandey: None. Supplements derived from plant extracts are often used as alternatives to pharmaceutical drugs. These products are often perceived as safe despite the absence of scientific evidence regarding their efficacy and safety and are less strictly regulated than pharmaceutical products. Winter cherry (Withania somnifera, commonly known as ashwagandha) is used as a testosterone booster supplement in multiple countries. Withania somnifera extract was found to increase testosterone and androgen precursors in a small clinical study. However, there is a lack of data regarding effects of Withania somnifera on the steroidogenic pathways. Steroidogenesis impacts not only sexual differentiation, reproduction, and fertility but also other physiological processes, including hypertension and obesity. Moreover, the modulation of hormone signaling is an important target in hormone-responsive pathogenesis, and overproduction of androgens has been implicated in the pathogenesis of prostate cancer (PCa) and polycystic ovarian syndrome (PCOS). In humans, testosterone is mainly produced in the testes after conversion of pregnenolone to 17-hydroxypregnenolone (17-OHPreg) and dehydroepiandrosterone (DHEA, the precursor of androgens) by adrenal CYP17A1, which catalyzes both the 17-hydroxylation of steroids for cortisol production and the breakage of 17,20 bonds (17,20 lyase) in 17-OHPreg to produce DHEA. The intake of substances that may increase androgen levels by people with PCa or PCOS or with predisposition to these conditions may trigger pathogenesis or worsen prognosis by accelerating disease progression. We investigated the effects of Withania somnifera in modulating the adrenal steroidogenic pathway and the possible consequences on the pathogenesis of androgen-responsive conditions, specifically PCa. Adrenal cells were treated with the extract of Withania somnifera to characterize its effect on adrenal steroid profile and CYP17A1 hydroxylase and lyase activities. Prostate cancer proliferation was assessed by direct incubation of prostate cancer cells with extracts as well as with conditioned media from adrenal cells treated for 48h, to simulate natural steroidogenesis. The results suggest that adrenal steroid hormone biosynthesis can be altered by the intake of Withania somnifera extract and that this supplement may affect proliferation of PCa cells. Moreover, the reported testosterone booster effect of Withania somnifera supplementation does not appear to be through increased adrenal androgen production. Presentation: 6/1/2024

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

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&amp;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

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

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&amp;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&amp;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&amp;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

A STAR for the ages: a 30-year historical perspective of the role of transcription factors in the regulation of steroidogenic acute regulatory gene expression.

The steroidogenic acute regulatory (STAR) protein is an essential cholesterol transporter that shuttles cholesterol from the outer to the inner mitochondrial membrane in the major steroidogenic endocrine organs. It is a key player in the acute regulation of steroid hormone biosynthesis in response to tropic hormone stimulation. Its discovery 30 years ago sparked immediate interest in understanding how STAR action is controlled. Since increased STAR gene expression is a classic feature of the acute regulation of steroidogenesis, a special emphasis was placed on defining the transcriptional regulatory mechanisms that underlie its rapid induction in response to tropic hormone stimulation. These mechanisms include the effects of enhancers, the regulation of chromatin accessibility, the impact of epigenetic factors, and the role of transcription factors. Over the past three decades, understanding the transcription factors that regulate STAR gene expression has been the subject of more than 170 independent scientific publications, making it one of, and if not the best, studied genes in the steroidogenic pathway. This intense research effort has led to the identification of dozens of transcription factors and their related binding sites in STAR 5' flanking (promoter) sequences across multiple species. STAR gene transcription appears to be complex in that a large number of transcription factors have been proposed to interact with either isolated or overlapping regulatory sequences that are tightly clustered over a relatively short promoter region upstream of the STAR transcription start site. Many of these transcription factors appear to work in unique combinatorial codes and are impacted by diverse hormonal and intracellular signaling pathways. This review provides a retrospective overview of the transcription factors proposed to regulate both basal and acute (hormonal) STAR gene expression, and how insights in this area have evolved over the past 30 years.

Psoriatic skin transcript phenotype: androgen/estrogen and cortisone/cortisol imbalance with increasing DNA damage response.

Patients prone to psoriasis suffer after a breakdown of the epidermal barrier and develop poorly healing lesions with abnormal proliferation of keratinocytes. Strong inflammatory reactions with genotoxicity (short telomeres) suggest impaired immune defenses with DNA damage repair response (DDR) in patients with psoriasis. Recent evidence indicates the existence of crosstalk mechanisms linking the DDR machinery and hormonal signaling pathways that cooperate to influence both progressions of many diseases and responses to treatment. The aim of this study was to clarify whether steroid biosynthesis and genomic stability markers are altered in parallel during the formation of psoriatic skin. Understanding the interaction of the steroid pathway and DNA damage response is crucial to addressing underlying fundamental issues and managing resulting epidermal barrier disruption in psoriasis. Skin (Lesional, non-lesional) and blood samples from twenty psoriasis patients and fifteen healthy volunteers were collected. Real-Time-PCR study was performed to assess levels of known transcripts such as: estrogen (ESR1, ESR2), androgen (AR), glucocorticoid/mineralocorticoid receptors (NR3C1, NR3C2), HSD11B1/HSD11B2, and DNA damage sensors (SMC1A, TREX1, TREX2, SSBP3, RAD1, RAD18, EXO1, POLH, HUS1). We found that ESR1, ESR2, HSD11B1, NR3C1, NR3C2, POLH, and SMC1A transcripts were significantly decreased and AR, TREX1, RAD1, and SSBP3 transcripts were increased dramatically in the lesional skin compared to skin samples of controls. We found that the regulation of the steroidogenic pathway was disrupted in the lesional tissue of psoriasis patients and that a sufficient glucocorticoid and mineralocorticoid response did not form and the estrogen/androgen balance was altered in favour of androgens. We suggest that an increased androgen response in the presence of DDR increases the risk of developing psoriasis. Although this situation may be the cause or the consequence of a disruption of the epidermal barrier, our data suggest developing new therapeutic strategies.

Inositol Restores Appropriate Steroidogenesis in PCOS Ovaries Both In Vitro and In Vivo Experimental Mouse Models.

Androgen excess is a key feature of several clinical phenotypes of polycystic ovary syndrome (PCOS). However, the presence of FSH receptor (FSHR) and aromatase (CYP19A1) activity responses to physiological endocrine stimuli play a critical role in the pathogenesis of PCOS. Preliminary data suggest that myo-Inositol (myo-Ins) and D-Chiro-Inositol (D-Chiro-Ins) may reactivate CYP19A1 activity. We investigated the steroidogenic pathway of Theca (TCs) and Granulosa cells (GCs) in an experimental model of murine PCOS induced in CD1 mice exposed for 10 weeks to a continuous light regimen. The effect of treatment with different combinations of myo-Ins and D-Chiro-Ins on the expression of Fshr, androgenic, and estrogenic enzymes was analyzed by real-time PCR in isolated TCs and GCs and in ovaries isolated from healthy and PCOS mice. Myo-Ins and D-Chiro-Ins, at a ratio of 40:1 at pharmacological and physiological concentrations, positively modulate the steroidogenic activity of TCs and the expression of Cyp19a1 and Fshr in GCs. Moreover, in vivo, inositols (40:1 ratio) significantly increase Cyp19a1 and Fshr. These changes in gene expression are mirrored by modifications in hormone levels in the serum of treated animals. Myo-Ins and D-Chiro-Ins in the 40:1 formula efficiently rescued PCOS features by up-regulating aromatase and FSHR levels while down-regulating androgen excesses produced by TCs.

TFEB controls syncytiotrophoblast formation and hormone production in placenta.

TFEB, a bHLH-leucine zipper transcription factor belonging to the MiT/TFE family, globally modulates cell metabolism by regulating autophagy and lysosomal functions. Remarkably, loss of TFEB in mice causes embryonic lethality due to severe defects in placentation associated with aberrant vascularization and resulting hypoxia. However, the molecular mechanism underlying this phenotype has remained elusive. By integrating in vivo analyses with multi-omics approaches and functional assays, we have uncovered an unprecedented function for TFEB in promoting the formation of a functional syncytiotrophoblast in the placenta. Our findings demonstrate that constitutive loss of TFEB in knock-out mice is associated with defective formation of the syncytiotrophoblast layer. Indeed, using in vitro models of syncytialization, we demonstrated that TFEB translocates into the nucleus during syncytiotrophoblast formation and binds to the promoters of crucial placental genes, including genes encoding fusogenic proteins (Syncytin-1 and Syncytin-2) and enzymes involved in steroidogenic pathways, such as CYP19A1, the rate-limiting enzyme for the synthesis of 17β-Estradiol (E2). Conversely, TFEB depletion impairs both syncytial fusion and endocrine properties of syncytiotrophoblast, as demonstrated by a significant decrease in the secretion of placental hormones and E2 production. Notably, restoration of TFEB expression resets syncytiotrophoblast identity. Our findings identify that TFEB controls placental development and function by orchestrating both the transcriptional program underlying trophoblast fusion and the acquisition of endocrine function, which are crucial for the bioenergetic requirements of embryonic development.

P-661 Comparative and descriptive molecular analysis of cumulus and luteinized mural granulosa cells for steroidogenic function in IVF patients

Abstract Study question Do cumulus granulosa cells (CCs) have steroidogenic function and gonadotropin response like luteinized mural granulosa cells (LMGCs)? Summary answer Yes. Both types of granulosa cells have similar expression levels for the steroidogenic enzymes and hCG/LH receptors. What is known already CCs and LMGCs have distinct roles in follicular development, oocyte growth and maturation, cumulus expansion, ovulation, and luteinization. CCs support oocytes, especially during meiotic resumption, ovulation, and fertilization. They exhibit specialized functions in response to hormones like LH and participate in bidirectional communication with the oocyte. Although LMGCs are primarily responsible for steroidogenic function and progesterone (P4) production, very limited data is available as to whether CC themselves express the steroidogenic enzymes and gonadotropin receptors and produce sex steroids at basal or after gonadotropin stimulation in humans. We aimed to address this issue in the current study. Study design, size, duration An in-vitro basic science study was designed to compare CCs and LGMCs in the context of vitality, steroidogenic capabilities (both in baseline and response to rec-hCG (10 IU/ml), and EGF pathway responses. The assessment was planned to compare hormonal assay in spent culture and transcriptional levels. The study’s duration encompassed the period of ovarian stimulation, oocyte retrieval, and subsequent cellular analysis. Participants/materials, setting, methods 24 IVF patients undergoing ovarian stimulation with progestin primed (PPOS) and GnRH antagonist protocols for different infertility etiologies were included. Ovarian stimulation and ovulation trigger were done using rec-FSH and rec-hCG, respectively. CCs were collected from the denudation plate. LMGCs were obtained from follicular aspirates during the OPU and cultured individually for each patient without pooling in culture plates. After overnight incubation, the cells were treated with rec-hCG overnight. RNA isolation was performed. Main results and the role of chance The expression of the steroidogenic enzymes (StAR, SCC, 17B-HSD, 3B-HSD, Aromatase), LH receptor and EGF-like amphiregulin (AREG) in CCs were comparable to LMGCs on quantitative RT-PCR analysis. In line with this finding, we found they produced similar amounts of estradiol (E2) and progesterone (P4) in culture. Furthermore, similar to LMGCs, hCG treatment of the CCs resulted in a significant increase in P4 output, indicative of the presence of functional LH receptors. When the culture period was extended to six days, CCs retained their vitality and steroidogenic function like LMGCs and produced detectable E2 and P4. Limitations, reasons for caution Caution should be exercised when interpreting our data as it is unclear if the parameters analyzed here vary according to the infertility etiology, stimulation protocol, mode of trigger and any underlying disease that concomitantly present. Wider implications of the findings Prominent steroidogenic activity of CCs might have other important implications for a successful pregnancy. Moreover, similar expression profiles of steroidogenic pathways and hCG/LH receptors between CCs and LMGCs indicate that CCs can also be used to understand better the luteal function and intricate crosstalk between in oocyte microenvironment. Trial registration number N/A

Patients with gynecological malignancies are similar to other IVF patients without cancer for clinical and molecular reproductive parameters and DNA damage response pattern

This study intended to investigate if gynecological cancers compromise ovarian function and reduce the success of assisted reproduction techniques (ART). No clinical and molecular data together is available on this issue for gynecological or other organ cancers. Steroidogenic pathways and DNA damage response characteristics of the granulosa cells retrieved from the 39 gynecological cancer patients were analyzed together with their clinical ART characteristics in comparison to 31 control ART patients. Patients with gynecological malignancies were similar to the control IVF patients for the number of mature oocytes retrieved, fertilization rates and embryo development competency. Molecular analyses of the granulosa cells retrieved from these cancer patients did not detect any perturbations in gonadotropin receptor expression and response, sex steroid production, cholesterol utilization/storage and, DNA damage response pattern in comparison to control IVF patients without cancer. This study provides the first reassuring clinical and molecular combined data set that the presence of gynecological malignancy does not appear to have any detrimental effect on clinical IVF cycle characteristics and ovarian functioning at molecular level.

High Salt Diet Regulates Intrarenal Sex Steroid Biosynthesis Pathway in a Sex-Specific Manner

Hypertension is more prevalent in men than age-matched pre-menopausal women. Sodium (Na+) homeostasis plays a major role in the control of blood pressure. Renal medullary estrogenic signaling promotes Na+ excretion in female, but not male Sprague Dawley (SD) rats implicating estrogen as a potential female-specific natriuretic factor. Recently we showed that the components of estrogen biosynthesis pathway are expressed in rat kidney suggesting the kidneys can produce estrogen locally to invoke this natriuretic response. It is unknown how dietary Na+ impacts biosynthesis of sex hormones. We hypothesize that a high salt (HS) diet enhances intrarenal estrogen biosynthesis pathway, and this enhancement is more pronounced in females compared to males. To test this hypothesis, we harvested the renal cortex from 14 week old male and female SD rats maintained on either a normal salt (NS 0.3% NaCl) or HS (4% NaCl) diet for 5 days. Given that cholesterol is the main precursor of steroid hormones, we measured the mRNA expression of HMG-CoA reductase (HMGCR), the key enzyme for cholesterol synthesis; and the steroidogenic acute regulatory protein (StAR), which transports cholesterol into the mitochondria for steroid commitment. We also assessed the mRNA expression of steroid 5α reductase (SRD5A), which converts testosterone to the non-aromatizable androgen, dihydrotestosterone. Further, we assessed aromatase, the key enzyme in estrogen biosynthesis, via immunohistochemistry. We found that renal HMGCR was more abundant in female compared to male rats maintained on a NS diet (P=0.0467). Increasing dietary salt intake upregulated renal HMGCR expression in male (P=0.0020), but not female rats (NS male; 100.0 ± 26.8 vs. NS female; 235.3 ± 23.2 vs. HS male 294.6 ± 42.0 vs HS female 314.6 ± 21.0 relative expression % NS male n=6-7/group). On a NS diet, renal StAR was more abundant in female compared to male rats (NS male; 100.0 ± 25.3 vs. NS female; 217.2 ± 36.5 relative expression % NS male P=0.0255 n=5-7/group). Increasing dietary salt did not alter expression of StAR in either sex. Similarly, renal SRD5A expression was higher in female compared to male (P=0.0141) rats. In addition, a HS diet increased the expression of SRD5A in female rats (0.0093) and tended to increase SRD5A expression in male (P=0.0849) rats (NS male; 100.0 ± 11.6 vs. NS female; 297.8 ± 37.7 vs. HS male 243.0 ± 39.2 vs HS female 498.8 ± 44.8 relative expression % NS male n=6-7/group). We also revealed that aromatase was expressed at a comparable level in male and female renal cortex, regardless of the diet (NS male; 2.4 ± 0.3 vs. NS female; 2.6 ± 0.3 vs. HS male 2.8 ± 0.3 vs HS female 1.9 ± 0.1 Average intensity x area mM2 n=6-9/group). Together, this data suggests that (i) the female kidney may be more primed for steroid biosynthesis and (ii) there is a sex-specific upregulation of the intrarenal sex steroid biosynthesis pathway in response to increasing dietary salt intake. Additional work is required to investigate the potential role of the kidney in androgen and estrogen production. A better understanding of this steroidogenic pathway could reveal novel sex-specific therapeutics for hypertension. T32DK007569-34 Carl Gottschalk Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of Curcuma longa extract on reproduction function in mice and testosterone production in Leydig cells.

Curcuma longa, best known for its culinary application as the main constituent of curry powder, has shown potential impact on the reproductive system. This study aimed to investigate the efficacy of Curcuma longa extract (CLE) on Kidney-Yang deficiency mice induced by hydrocortisone and the possible roles in testosterone secretion in Leydig cells. We evaluated male sexual behaviour, reproductive organ weight, testosterone levels, and histological tissue changes in hydrocortisone-induced mice. CLE effectively reversed hydrocortisone-induced Kidney-Yang deficiency syndrome by improving sexual behaviour, testis and epididymis weight, testosterone levels and reducing pathological damage. Our invitro study further indicated that CLE stimulated testosterone production via upregulating the mRNA and protein expression of steroidogenic enzymes in Leydig cells. It significantly improved H89-inhibited protein expression of StAR and cAMP-response element-binding (CREB), as well as melatonin-suppressed StAR protein expression. The data obtained from this study suggest that CLE could alleviate Kidney-Yang deficiency symptoms and stimulate testosterone production by upregulating the steroidogenic pathway. This research identifies CLE as a potential nutraceutical option for addressing testosterone deficiency diseases.

Genistein effect in cultured ovine ovarian granulosa cells.

Genistein, an isoflavone has the potential to mimic, augment, or dysregulate the steroid hormone production pathways. We hypothesized that genistein affects the granulosa cell (GCs) functions through a series of biochemical, molecular, and genomic cascades. The present study was conducted to evaluate the impact of genistein exposure on GCs viability, apoptosis, and steroidogenesis. The present study involved 3/5 days of exposure to genistein on GCs collected from abattoir-derived ovine ovaries at doses of 0, 1, 10, 25, 50, and 100 µM. The harvested GCs were used for growth, cytotoxicity, and gene expression studies related to apoptosis, growth, and steroidogenesis. We observed that genistein had both stimulatory at 10 and 25 µM levels as well as inhibitory effects at 50 and 100 µM levels on the growth and proliferation of GCs. Genistein significantly decreased the levels of 17β-estradiol at higher exposure (50 and 100 µM), whereas the progesterone level increased significantly as the genistein exposure increased. Additionally, genistein could also alter the mRNA expression of the steroidogenic receptor, enzymes, proteins, and growth-related genes suggesting that genistein could potentially alter the steroidogenic pathways. We conclude that genistein can interfere with cell survival and steroidogenesis by exhibiting a dose-dependent biphasic response on the viability, growth-related parameters, and the synthesis of 17β-estradiol in the cultured GCs.

Aging induces region-specific dysregulation of hormone synthesis in the primate adrenal gland.

Adrenal glands, vital for steroid secretion and the regulation of metabolism, stress responses and immune activation, experience age-related decline, impacting systemic health. However, the regulatory mechanisms underlying adrenal aging remain largely uninvestigated. Here we established a single-nucleus transcriptomic atlas of both young and aged primate suprarenal glands, identifying lipid metabolism and steroidogenic pathways as core processes impacted by aging. We found dysregulation in centripetal adrenocortical differentiation in aged adrenal tissues and cells in the zona reticularis region, responsible for producing dehydroepiandrosterone sulfate (DHEA-S), were highly susceptible to aging, reflected by senescence, exhaustion and disturbed hormone production. Remarkably, LDLR was downregulated in all cell types of the outer cortex, and its targeted inactivation in human adrenal cells compromised cholesterol uptake and secretion of dehydroepiandrosterone sulfate, as observed in aged primate adrenal glands. Our study provides crucial insights into endocrine physiology, holding therapeutic promise for addressing aging-related adrenal insufficiency and delaying systemic aging.