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Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed lists a range of substances from botanical origin (weed seeds) and additionally some chemical compounds directly originating from specific weeds. In order to examine the actual status of enforcement and of the present occurrence of these botanical substances, a survey was carried out. A questionnaire was sent to 103 laboratories, including official control labs from all member states of the European Union. The results, indicating the frequency of occurrence as far as reported, are compared to the publications of the EU Rapid Alert System for Food and Feed (RASFF). A total of 44 questionnaires was returned (42.7%) from 22 member states. Ten member states predominantly from north-western Europe appeared to have an active monitoring of botanical undesirable substances. The questionnaire results did not indicate that the other member states enforce this part of Directive 2002/32/EC. Reports on the frequency of occurrence include: a few to 25-50% of the samples contain traces of ergot (8 member states), a few to 24% contain at least some traces of thorn apple (6 member states), zero to 17% contain some castor oil plant seeds (3 member states), zero to a few samples contain Crotalaria seeds (3 member states), and zero to 6% contain traces of Sareptian mustard (4 member states). One member state conducted extra surveillance since several cases of animal intoxications have been reported. In some cases a coincidence with undesirable botanical substances was found.

We examined the effect of the puberty blocker, leuprolide acetate, on sex differences in juvenile rough-and-tumble play behavior and anxiety-like behavior in adolescent male and female rats. We also evaluated leuprolide treatment on gonadal and pituitary hormone levels and activity-regulated cytoskeleton-protein messenger RNA levels within the adolescent amygdala, a region important both for rough-and-tumble play and anxiety-like behavior. Our findings suggest that leuprolide treatment lowered anxiety-like behavior during adolescent development, suggesting that the maturation of gonadotropin-releasing hormone systems may be linked to increased anxiety. These data provide a potential new model to understand the emergence of increased anxiety triggered around puberty. Leuprolide also reduced masculinized levels of rough-and-tumble play behavior, lowered follicle-stimulating hormone, and produced a consistent pattern of reducing or halting sex differences of hormone levels, including testosterone, growth hormone, thyrotropin, and corticosterone levels. Therefore, leuprolide treatment not only pauses sexual development of peripheral tissues, but also reduces sex differences in hormones, brain, and behavior, allowing for better harmonization of these systems following gender-affirming hormone treatment. These data contribute to the intended use of puberty blockers in stopping sex differences from developing further with the potential benefit of lowering anxiety-like behavior., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

During male fetal development, testosterone plays an essential role in the differentiation and maturation of the male reproductive system. Deficient fetal testosterone production can result in variations of sex differentiation that may cause infertility and even increased tumor incidence later in life. Fetal Leydig cells in the fetal testis are the major androgen source in mammals. Although fetal and adult Leydig cells are similar in their functions, they are two distinct cell types, and therefore, the knowledge of adult Leydig cells cannot be directly applied to understanding fetal Leydig cells. This review summarizes our current knowledge of fetal Leydig cells regarding their cell biology, developmental biology, and androgen production regulation in rodents and human. Fetal Leydig cells are present in basement membrane-enclosed clusters in between testis cords. They originate from the mesonephros mesenchyme and the coelomic epithelium and start to differentiate upon receiving a Desert Hedgehog signal from Sertoli cells or being released from a NOTCH signal from endothelial cells. Mature fetal Leydig cells produce androgens. Human fetal Leydig cell steroidogenesis is LHCGR (Luteinizing Hormone Chronic Gonadotropin Receptor) dependent, while rodents are not, although other Gα s -protein coupled receptors might be involved in rodent steroidogenesis regulation. Fetal steroidogenesis ceases after sex differentiation is completed, and some fetal Leydig cells dedifferentiate to serve as stem cells for adult testicular cell types. Significant gaps are acknowledged: (1) Why are adult and fetal Leydig cells different? (2) What are bona fide progenitor and fetal Leydig cell markers? (3) Which signaling pathways and transcription factors regulate fetal Leydig cell steroidogenesis? It is critical to discover answers to these questions so that we can understand vulnerable targets in fetal Leydig cells and the mechanisms for androgen production that when disrupted, leads to variations in sex differentiation that range from subtle to complete sex reversal., (© 2024 The Authors. Molecular Reproduction and Development published by Wiley Periodicals LLC.)

Background: Preeclampsia shares numerous risk factors with cardiovascular diseases. Here, we aimed to assess the potential utility of high-sensitivity cardiac troponin I (hs-cTnI) values during pregnancy in predicting preeclampsia occurrence., Methods: This study measured hs-cTnI levels in 3721 blood samples of 2245 pregnant women from 4 international, prospective cohorts. Three analytical approaches were used: (1) a cross-sectional analysis of all women using a single blood sample, (2) a longitudinal analysis of hs-cTnI trajectories in women with multiple samples, and (3) analyses of prediction models incorporating hs-cTnI, maternal factors, and the sFlt-1 (soluble fms-like tyrosine kinase 1)/PlGF (placental growth factor) ratio., Results: Women with hs-cTnI levels in the upper quarter had higher odds ratios for preeclampsia occurrence compared with women with levels in the lower quarter. Associations were driven by preterm preeclampsia (odds ratio, 5.78 [95% CI, 2.73-12.26]) and remained significant when using hs-cTnI as a continuous variable adjusted for confounders. Between-trimester hs-cTnI trajectories were independent of subsequent preeclampsia occurrence. A prediction model incorporating a practical hs-cTnI level of detection cutoff (≥1.9 pg/mL) alongside maternal factors provided comparable performance with the sFlt-1/PlGF ratio. A comprehensive model including sFlt-1/PlGF, maternal factors, and hs-cTnI provided added value (cross-validated area under the receiver operator characteristic, 0.78 [95% CI, 0.73-0.82]) above the sFlt-1/PlGF ratio alone (cross-validated area under the receiver operator characteristic, 0.70 [95% CI, 0.65-0.76]; P =0.027). As assessed by likelihood ratio tests, the addition of hs-cTnI to each prediction model significantly improved the respective prediction model not incorporating hs-cTnI, particularly for preterm preeclampsia. Net reclassification improvement analyses indicated that incorporating hs-cTnI improved risk prediction predominantly by correctly reclassifying women with subsequent preeclampsia occurrence., Conclusions: These exploratory findings uncover a potential role for hs-cTnI as a complementary biomarker in the prediction of preeclampsia. After validation in prospective studies, hs-cTnI, alongside maternal factors, may either be considered as a substitute for angiogenic biomarkers in health care systems where they are sparce or unavailable, or as an enhancement to established prediction models using angiogenic markers., Competing Interests: Disclosures sFlt-1 and PlGF in OCC were initially measured in collaboration with BRAHMS GmbH at their facility in Hennigsdorf, Berlin. For this study, BRAHMS had no further involvement in collection or interpretation of data, writing of the report, or decision to submit the article for publication. S.B. has received research funding from Abbott, Abbott Diagnostics, Bayer, Boehringer Ingelheim, Siemens, and Thermo Fisher; honoraria for lectures from Abbott, Abbott Diagnostics, AstraZeneca, Bayer, Boehringer Ingelheim, Medtronic, Pfizer, Roche, Siemens Diagnostics, Siemens, and Thermo Fisher; and honoraria for advisory board memberships and consulting from Boehringer Ingelheim, Bayer, Novartis, Roche, and Thermo Fisher. T.Z. and S.B. are listed as coinventors of an international patent on the use of a computing device to estimate the probability of myocardial infarction (International Publication No. WO2022043229A1). T.Z. is a shareholder in ART.EMIS GmbH Hamburg. The other authors report no conflicts.

Appropriate embryo-uterine interactions are essential for implantation. Besides oocyte abnormalities, implantation failure is a major contributor to early pregnancy loss. Previously, we demonstrated that two members of the Iroquois homeobox transcription factor family, IRX3 and IRX5, exhibited distinct and dynamic expression profiles in the developing ovary to promote oocyte and follicle survival. Elimination of each gene independently caused subfertility, but with different breeding pattern outcomes. Irx3 KO (Irx3LacZ/LacZ) females produced fewer pups throughout their reproductive lifespan which could only be partially explained by poor oocyte quality. Thus, we hypothesized that IRX3 is also expressed in the uterus where it acts to support pregnancy. To test this hypothesis, we harvested pregnant uteri from control and Irx3 KO females to evaluate IRX3 expression profiles and the integrity of embryo implantation sites. Our results indicate that IRX3 is expressed in the endometrial stromal cells at day 4 of pregnancy (D4) with peak expression at D5-D6, and then greatly diminishes by D7. Further, studies showed that while embryos were able to attach to the uterus, implantation sites in Irx3 KO pregnant mice exhibited impaired vascularization and abnormal expression of decidualization markers. Finally, we also observed an impaired response of the Irx3 KO uteri to an artificial deciduogenic stimulus, indicating a critical role of this factor in regulating the decidualization program. Together, these data established that IRX3 promotes female fertility via at least two different mechanisms: (1) promoting competent oocytes and (2) facilitating functional embryo-uterine interactions during implantation., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

STARD1 stimulates cholesterol transfer to mitochondrial CYP11A1 for conversion to pregnenolone. A cholesterol-binding START domain is guided by an N-terminal domain in a cell selective manner. Fetal and adult Leydig cells (FLC, ALC) show distinct Stard1 regulation. sm- FISH microscopy, which resolves individual molecules of Stard1 mRNA, shows uniformly high basal expression in each FLC. In ALC, in vivo, and cultured MA-10 cells, basal Stard1 expression is minimal. PKA activates loci asynchronously, with delayed splicing/export of 3.5 kb mRNA to mitochondria. After 60 min, ALC transition to an integrated mRNA delivery to mitochondria that is seen in FLC. Sertoli cells cooperate in Stard1 stimulation in FLC by delivering DHH to the primary cilium. There PTCH, SMO and cholesterol cooperate to release GLI3 to activate the Stard1 locus, probably by directing histone changes. ALC lack cilia. PKA then primes locus activation. FLC and ALC share similar SIK/CRTC/CREB regulation characterized for adrenal cells., (Copyright © 2021. Published by Elsevier B.V.)

Two specialized functions of cholesterol during fetal development include serving as a precursor to androgen synthesis and supporting hedgehog (HH) signaling activity. Androgens are produced by the testes to facilitate masculinization of the fetus. Recent evidence shows that intricate interactions between the HH and androgen signaling pathways are required for optimal male sex differentiation and defects of either can cause birth anomalies indicative of 46,XY male variations of sex development (VSD). Further, perturbations in cholesterol synthesis can cause developmental defects, including VSD, that phenocopy those caused by disrupted androgen or HH signaling, highlighting the functional role of cholesterol in promoting male sex differentiation. In this review, we focus on the role of cholesterol in systemic androgen and local HH signaling events during fetal masculinization and their collective contributions to pediatric VSD., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

STARD1 moves cholesterol (CHOL) from the outer mitochondrial membrane (OMM) to the inner membrane (IMM) in steroidogenic cells. This activity is integrated into CHOL trafficking and synthesis homeostasis, involving uptake through SR-B1 and LDL receptors and distribution through endosomes, ER, and lipid droplets. In adrenal cells, STARD1 is imported into the mitochondrial matrix accompanied by delivery of several hundred CHOL molecules. This transfer limits CYP11A1-mediated generation of pregnenolone. CHOL transfer is coupled to translation of STARD1 mRNA at the OMM. In testis cells, slower CHOL trafficking seems to be limiting. STARD1 also functions in a slower process through ER OMM contacts. The START domain of STARD1 is utilized by a family of genes, which includes additional STARD (forms 3-6) and GRAMD1B proteins that transfer CHOL. STARD forms 2 and 7 deliver phosphatidylcholine. STARD1 and STARD7 target their respective activities to mitochondria, via N-terminal domains (NTD) of over 50 amino acids. The NTD is not essential for steroidogenesis but exerts tissue-selective enhancement (testis>>adrenal). Three conserved sites for cleavage by the mitochondrial processing protease (MPP) generate three forms, each potentially with specific functions, as demonstrated in STARD7. STARD1 is expressed in macrophage and cardiac repair fibroblasts. Additional functions include CHOL metabolism by CYP27A1 that directs activation of LXR and CHOL export processes. STARD1 generates 3.5- and 1.6-kb mRNA from alternative polyadenylation. The 3.5-kb form exclusively binds the PKA-induced regulator, TIS11b, which binds at conserved sites in the extended 3'UTR to control mRNA translation and turnover. STARD1 expression also exhibits a novel, slow splicing that delayed splicing delivery of mRNA to mitochondria. Stimulation of transcription by PKA is directed by suppression of SIK forms that activate a CRTC/CREB/CBP promoter complex. This process is critical to pulsatile hormonal activation in vivo . sm-FISH RNA imaging shows a flow of single STARD1 mRNA particles from asymmetric accumulations of primary transcripts at gene loci to 1:1 complex of 3.5-kb mRNA with peri-nuclear mitochondria. Adrenal cells are similar but distinguished from testis cells by appreciable basal expression prior to hormonal activation. This difference is conserved in culture and in vivo ., (Copyright © 2020 Larsen, Lee, Jorgensen and Jefcoate.)

Healthy development of ovarian follicles depends on appropriate interactions and function between oocytes and their surrounding granulosa cells. Previously, we showed that double knockout of Irx3 and Irx5 (Irx3/5 DKO) in mice resulted in abnormal follicle morphology and follicle death. Further, female mouse models of individual Irx3 or Irx5 knockouts were both subfertile but with distinct defects. Notably, the expression profile of each gene suggests independent roles for each; first, they are colocalized in pre-granulosa cells during development that then progresses to include oocyte expression during germline nest breakdown and primordial follicle formation. Thereafter, their expression patterns diverge between oocytes and granulosa cells coinciding with the formulation and maturation of intimate oocyte-granulosa cell interactions. The objective of this study was to investigate the contributions of Irx5 and somatic cell-specific expression of Irx3 during ovarian development. Our results show that Irx3 and Irx5 contribute to female fertility through different mechanisms and that Irx3 expression in somatic cells is important for oocyte quality and survival. Based on evaluation of a series of genetically modified mouse models, we conclude that IRX3 and IRX5 collaborate in the same cells and then in neighboring cells to foster a healthy and responsive follicle. Long after these two factors have extinguished, their legacy enables these intercellular connections to mature and respond to extracellular signals to promote follicle maturation and ovulation., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Urogenital tract abnormalities are among the most common congenital defects in humans. Male urogenital development requires Hedgehog-GLI signaling and testicular hormones, but how these pathways interact is unclear. We found that Gli3XtJ mutant mice exhibit cryptorchidism and hypospadias due to local effects of GLI3 loss and systemic effects of testicular hormone deficiency. Fetal Leydig cells, the sole source of these hormones in developing testis, were reduced in numbers in Gli3XtJ testes, and their functional identity diminished over time. Androgen supplementation partially rescued testicular descent but not hypospadias in Gli3XtJ mutants, decoupling local effects of GLI3 loss from systemic effects of androgen insufficiency. Reintroduction of GLI3 activator (GLI3A) into Gli3XtJ testes restored expression of Hedgehog pathway and steroidogenic genes. Together, our results show a novel function for the activated form of GLI3 that translates Hedgehog signals to reinforce fetal Leydig cell identity and stimulate timely INSL3 and testosterone synthesis in the developing testis. In turn, exquisite timing and concentrations of testosterone are required to work alongside local GLI3 activity to control development of a functionally integrated male urogenital tract., Competing Interests: The authors have declared that no competing interests exist.

Members of the Iroquois B (IrxB) homeodomain cluster genes, specifically Irx3 and Irx5 , are crucial for heart, limb and bone development. Recently, we reported their importance for oocyte and follicle survival within the developing ovary. Irx3 and Irx5 expression begins after sex determination in the ovary but remains absent in the fetal testis. Mutually antagonistic molecular signals ensure ovary versus testis differentiation with canonical Wnt/β-catenin signals paramount for promoting the ovary pathway. Notably, few direct downstream targets have been identified. We report that Wnt/β-catenin signaling directly stimulates Irx3 and Irx5 transcription in the developing ovary. Using in silico analysis of ATAC- and ChIP-Seq databases in conjunction with mouse gonad explant transfection assays, we identified TCF/LEF-binding sequences within two distal enhancers of the IrxB locus that promote β-catenin-responsive ovary expression. Meanwhile, Irx3 and Irx5 transcription is suppressed within the developing testis by the presence of H3K27me3 on these same sites. Thus, we resolved sexually dimorphic regulation of Irx3 and Irx5 via epigenetic and β-catenin transcriptional control where their ovarian presence promotes oocyte and follicle survival vital for future ovarian health., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Open microfluidic cell culture systems are powerful tools for interrogating biological mechanisms. We have previously presented a microscale cell culture system, based on spontaneous capillary flow of biocompatible hydrogels, that is integrated into a standard cell culture well plate, with flexible cell compartment geometries and easy pipet access. Here, we present two new injection molded open microfluidic devices that also easily insert into standard cell culture well plates and standard culture workflows, allowing seamless adoption by biomedical researchers. These platforms allow culture and study of soluble factor communication among multiple cell types, and the microscale dimensions are well-suited for rare primary cells. Unique advances include optimized evaporation control within the well, manufacture with reproducible and cost-effective rapid injection molding, and compatibility with sample preparation workflows for high resolution microscopy (following well-established coverslip mounting procedures). In this work, we present several use cases that highlight the usability and widespread utility of our platform including culture of limited primary testis cells from surgical patients, microscopy readouts including immunocytochemistry and single molecule fluorescence in situ hybridization (smFISH), and coculture to study interactions between adipocytes and prostate cancer cells.

Women and other mammalian females are born with a finite supply of oocytes that determine their reproductive lifespan. During fetal development, individual oocytes are enclosed by a protective layer of granulosa cells to form primordial follicles that will grow, mature, and eventually release the oocyte for potential fertilization. Despite the knowledge that follicles are dysfunctional and will die without granulosa cell-oocyte interactions, the mechanisms by which these cells establish communication is unknown. We previously identified that two members of the Iroquois homeobox transcription factor gene family, Irx3 and Irx5, are expressed within developing ovaries but not testes. Deletion of both factors (Irx3-Irx5EGFP/Irx3-Irx5EGFP) disrupted granulosa cell-oocyte contact during early follicle development leading to oocyte death. Thus, we hypothesized that Irx3 and Irx5 are required to develop cell-cell communication networks to maintain follicle integrity and female fertility. A series of Irx3 and Irx5 mutant mouse models were generated to assess roles for each factor. While both Irx3 and Irx5 single mutant females were subfertile, their breeding outcomes and ovary histology indicated distinct causes. Careful analysis of Irx3- and Irx5-reporter mice linked the cause of this disparity to dynamic spatio-temporal changes in their expression patterns. Both factors marked the progenitor pre-granulosa cell population in fetal ovaries. At the critical phase of germline nest breakdown and primordial follicle formation however, Irx3 and Irx5 transitioned to oocyte- and granulosa cell-specific expression respectively. Further investigation into the cause of follicle death in Irx3-Irx5EGFP/Irx3-Irx5EGFP ovaries uncovered specific defects in both granulosa cells and oocytes. Granulosa cell defects included poor contributions to basement membrane deposition and mis-localization of gap junction proteins. Granulosa cells and oocytes both presented fewer cell projections resulting in compromised cell-cell communication. Altogether, we conclude that Irx3 and Irx5 first work together to define the pregranulosa cell population of germline nests. During primordial follicle formation, they transition to oocyte- and granulosa cell-specific expression patterns where they cooperate in neighboring cells to build the foundation for follicle integrity. This foundation is left as their legacy of the essential oocyte-granulosa cell communication network that ensures and ultimately optimizes the integrity of the ovarian reserve and therefore, the female reproductive lifespan., Competing Interests: The authors have declared that no competing interests exist.

Equine carpal sheath effusion has multiple etiologies. The purpose of this retrospective study was to describe the prevalence of distinct musculoskeletal lesions lameness in a sample of horses with a clinical diagnosis of carpal sheath effusion. A total of 121 horses met inclusion criteria. Seventy-four percent (89/121) of horses were lame at presentation; middle-aged (9-18 years, 80%) and older (> 18 years, 85%) horses were lame more frequently than young horses (< 9 years, 44%). Ninety-three percent (113/121) were diagnosed with osseous and/or soft tissue abnormalities. Of these 113 horses, 10 exhibited osseous abnormalities, whereas 111 were diagnosed with soft tissue lesions. Eighty-four percent (93/111) of the soft tissue injuries extended from the caudodistal antebrachium to the palmar metacarpus. The superficial digital flexor tendon (98/111; 88%) and accessory ligament of the superficial digital flexor tendon (64/111; 58%) were the most commonly injured structures, with both structures affected in 41 (41/111; 37%) horses. Injuries within the caudodistal antebrachium included the superficial digital flexor musculotendinous junction (66), the accessory ligament of the superficial digital flexor tendon (64), and deep digital flexor muscle (21), in isolation or in combination with other structures. Increased echogenicity in the medial superficial digital flexor musculotendinous junction was detected in 40 horses and was significantly associated with increasing age (middle-aged, 19/40; old, 18/40). Findings from this study indicated that age should be taken into consideration for horses presented with carpal sheath effusion and that adjacent structures within the caudodistal antebrachium should be included in evaluations., (© 2015 American College of Veterinary Radiology.)

Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3-5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture., (© 2014 by the Society for the Study of Reproduction, Inc.)

The dependence of prostate cancer on androgens provides a targeted means of treating advanced disease. Unfortunately, androgen deprivation therapies eventually become ineffective, leading to deadly castration-resistant prostate cancer (CRPC). One of many factors implicated in the transition to CRPC is the onset of de novo steroidogenesis. Although reactivation of steroid receptors likely plays a pivotal role in aggressive CRPC, little is understood regarding the mechanisms whereby prostate cancer cells initiate and maintain steroidogenesis. We hypothesize that steroidogenic factor 1 (SF1, NR5A1, AD4BP), a key regulator of steroidogenesis in normal endocrine tissues, is expressed in CRPC where it stimulates aberrant steroidogenesis and fuels aggressive growth. Notably, SF1 is not expressed in normal prostate tissue. Our results indicated that SF1 was absent in benign cells but present in aggressive prostate cancer cell lines. Introduction of ectopic SF1 expression in benign human prostate epithelial cells (BPH-1) stimulated increased steroidogenic enzyme expression, steroid synthesis, and cell proliferation. In contrast, data from an aggressive human prostate cancer cell line (BCaPT10) demonstrated that SF1 was required for steroid-mediated cell growth because BCaPT10 cell growth was diminished by abiraterone treatment and short hairpin RNA-mediated knockdown of SF1 (shSF1). SF1-depleted cells also exhibited defective centrosome homeostasis. Finally, whereas xenograft experiments in castrated hosts with BCaPT10 control transplants grew large, invasive tumors, BCaPT10-shSF1 knockdown transplants failed to grow. Therefore, we conclude that SF1 stimulates steroid accumulation and controls centrosome homeostasis to mediate aggressive prostate cancer cell growth within a castrate environment. These findings present a new molecular mechanism and therapeutic target for deadly CRPC.

The ovary functions to chaperone the most precious cargo for female individuals, the oocyte, thereby allowing the passage of genetic material to subsequent generations. Within the ovary, single oocytes are surrounded by a legion of granulosa cells inside each follicle. These two cell types depend upon one another to support follicle formation and oocyte survival. The infrastructure and events that work together to ultimately form these functional follicles within the ovary are unprecedented, given that the oocyte originates as a cell like all other neighboring cells within the embryo prior to gastrulation. This review discusses the journey of the germ cell in the context of the developing female mouse embryo, with a focus on specific signaling events and cell-cell interactions that escort the primordial germ cell as it is specified into the germ cell fate, migrates through the hindgut into the gonad, differentiates into an oocyte, and culminates upon formation of the primordial and then primary follicle., (© 2013 Wiley Periodicals, Inc.)

MicroRNAs are important regulators of developmental gene expression, but their contribution to fetal gonad development is not well understood. We have identified the evolutionarily conserved gonadal microRNAs miR-202-5p and miR-202-3p as having a potential role in regulating mouse embryonic gonad differentiation. These microRNAs are expressed in a sexually dimorphic pattern as the primordial XY gonad differentiates into a testis, with strong expression in Sertoli cells. In vivo, ectopic expression of pri-miR-202 in XX gonads did not result in molecular changes to the ovarian determination pathway. Expression of the primary transcript of miR-202-5p/3p remained low in XY gonads in a conditional Sox9-null mouse model, suggesting that pri-miR-202 transcription is downstream of SOX9, a transcription factor that is both necessary and sufficient for male sex determination. We identified the pri-miR-202 promoter that is sufficient to drive expression in XY but not XX fetal gonads ex vivo. Mutation of SOX9 and SF1 binding sites reduced ex vivo transactivation of the pri-miR-202 promoter, demonstrating that pri-miR-202 may be a direct transcriptional target of SOX9/SF1 during testis differentiation. Our findings indicate that expression of the conserved gonad microRNA, miR-202-5p/3p, is downstream of the testis-determining factor SOX9, suggesting an early role in testis development.

These three case reports describe the long-term duration of function of ovarian cortical tissue grafts among patients in a university fertility preservation programme in Europe and in a private practice programme in the USA. One woman underwent sterilizing cancer treatment and had frozen ovarian tissue transplanted, and two women underwent fresh ovarian tissue transplants. The function of ovarian cortical strips has continued for more than 7 years in these three women, with the birth of eight healthy babies following a single graft per patient. In addition to these three cases, transplantation (repeatedly in some cases) of cryopreserved ovarian tissue has restored reproductive function to all other women in the study centres' programmes for some years. The sustained longevity of function of the transplanted tissue suggests that it may also be possible to postpone the normal time of menopause or to alleviate its symptoms., (Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Ovarian development absolutely depends on communication between somatic and germ cell components. In contrast, it is not until after birth that interactions between somatic and germ cells play an important role in testicular maturation and spermatogenesis. Previously, we discovered that Irx3 expression was localized specifically to female gonads during embryonic development; therefore, we sought to determine the function of this genetic locus in developing gonads of both sexes. The fused toes (Ft) mutant mouse is missing 1.6 Mb of chromosome 8, which includes the entire IrxB cluster (Irx3, Irx5, Irx6), Ftm, Fts, and Fto genes. Homozygote Ft mutant embryos die around embryonic day 13.5 (E13.5); therefore, to assess later development, we harvested gonads at E11.5 and transplanted them into nude mouse hosts. Our results show defects in somatic and germ cell maturation in developing gonads of both sexes. Testis development was normal initially; however, by 3-wk posttransplantation, expression of Sertoli and peritubular myoid cell markers were decreased. In many cases, gonocytes failed to migrate to structurally impaired basement membranes of seminiferous cords. Developmental abnormalities of the ovary appeared earlier and were more severe. Over time, the Ft mutant ovary formed very few primordial or primary follicles, which contained oocytes that failed to grow and were surrounded by scarce granulosa cells that expressed low levels of FOXL2. By 3 wk after transplantation, it was difficult to identify ovarian tissue in Ft mutant ovary transplants. In summary, we conclude that the Ft locus contains genes essential for somatic-germ cell interactions, without which the germ cell niche fails to mature in both sexes.

Targets of steroidogenic factor 1 (SF1; also known as NR5A1 and AD4BP) have been identified within cells at every level of the hypothalamic-pituitary-gonadal and -adrenal axes, revealing SF1 to be a master regulator of major endocrine systems. Mouse embryos express SF1 in the genital ridge until Embryonic Day 13.5 (E13.5). Thereafter, expression persists in the male and is substantially lower in the female gonad until birth. We hypothesize that the sexually dimorphic expression of Sf1 during gonadogenesis is mediated by sex-specific regulation of its promoter. To investigate dimorphic regulation within the fetal gonad, we developed an experimental strategy using transient transfection of E13.5 gonad explant cultures and evaluated various Sf1 promoter constructs for sexually dimorphic DNA elements. The proximal Sf1 promoter correctly targeted reporter activity to SF1-expressing cells in both XY and XX gonads. Stepwise deletion of sequences from the Sf1 promoter revealed two regions that affected regulation within female gonads. Mutation of both sequences together did not cause further disruption of reporter activity, suggesting the two sites might work in concert to promote activity in female somatic cells. Results from gel mobility shift assays and fetal gonad-chromatin immunoprecipitation showed that TCFAP2 binds to one of the two female-specific sites within the proximal promoter of Sf1. Together, we show that transient transfection experiments performed within developing testes and ovaries are a powerful tool to uncover elements within the Sf1 promoter that contribute to sex-specific expression.

Over the first 4 days of their life, primordial germ cells invade the endoderm, migrate into and through the developing hindgut, and traverse to the genital ridge where they cluster and ultimately inhabit the nascent gonad. Specific signal-receptor combinations between primordial germ cells and their immediate environment establish successful migration and colonization. Here we demonstrate that disruption of a cluster of six genes on murine chromosome 8, as exemplified by the Fused Toes (Ft) mutant mouse model, results in severely decreased numbers of primordial germ cells within the early gonad. Primordial germ cell migration appeared normal within Ft mutant embryos; however, germ cell counts progressively decreased during this time. Although no difference in apoptosis was detected, we report a critical decrease in primordial germ cell proliferation by E12.5. The six genes within the Ft locus include the IrxB cluster (Irx3, -5, -6), Fts, Ftm, and Fto, of which only Ftm, Fto, and Fts are expressed in primordial germ cells of the early gonad. From these studies, we have discovered that the Ft locus on mouse chromosome 8 is associated with cell cycle deficits within the primordial germ cell population that initiates just before translocation into the genital ridge., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Proper cell fate determination in mammalian gonads is critical for the establishment of sexual identity. The Hedgehog (Hh) pathway has been implicated in cell fate decision for various organs, including gonads. Desert Hedgehog (Dhh), one of the three mammalian Hh genes, has been implicated with other genes in the establishment of mouse fetal Leydig cells. To investigate whether Hh alone is sufficient to induce fetal Leydig cell differentiation, we ectopically activated the Hh pathway in Steroidogenic factor 1 (SF1)-positive somatic cell precursors of fetal ovaries. Hh activation transformed SF1-positive somatic ovarian cells into functional fetal Leydig cells. These ectopic fetal Leydig cells produced androgens and insulin-like growth factor 3 (INLS3) that cause virilization of female embryos and ovarian descent. However, the female reproductive system remained intact, indicating a typical example of female pseudohermaphroditism. The appearance of fetal Leydig cells was a direct consequence of Hh activation as evident by the absence of other testicular components in the affected ovary. This study provides not only insights into mechanisms of cell lineage specification in gonads, but also a model to understand defects in sexual differentiation.

Irx3 is a member of the Iroquois homeobox gene family that encodes a protein known for its essential role in spinal cord development. Transcript screening of male and female gonads during the critical period of sex determination (E12-13.5) revealed a sexually dimorphic expression pattern for Irx3 with female gonads exhibiting a sixfold increase in expression over time. Whole mount in situ hybridization confirmed the sexually dimorphic nature of Irx3 expression and immunohistochemical analysis of gonads at E13.5 determined that IRX3 and GATA4 proteins co-localized to somatic cells of XX gonads. The Irx3 signal persisted in germ cell-depleted XX gonads resulting from Busulfan treatment suggesting that its expression was independent of germ cell regulation. Quantitative real-time PCR analysis over an extended time course determined that Irx3 message was low initially and then increased in XX gonads until E13.5, remained elevated until birth, diminished shortly after birth, and remained low in the adult ovary. In contrast, Irx3 message was 50% lower in male compared to female gonads at the initial time point, and continued to decrease over time. Further analysis of adult ovaries suggested that IRX3 expression is not present in any subpopulations of cells of the differentiated ovary. Together, these results suggest that the Irx3 signal is restricted to the somatic cell component of XX gonads and is present at a discreet period of ovarian development that ends abruptly at birth. This timing coincides with the transition of female primordial germ cells from mitotic proliferation to meiotic division, and the organization of germ cell cysts prior to primordial follicle development at birth.

Normal reproductive function in mammals requires precise control of LH synthesis and secretion by gonadotropes of the anterior pituitary. Synthesis of LH requires expression of two genes [alpha-glycoprotein subunit (alphaGSU) and LHbeta] located on different chromosomes. Hormones from the hypothalamus and gonads modulate transcription of both genes as well as secretion of the biologically active LH heterodimer. In males and females, the transcriptional tone of the genes encoding alphaGSU and LHbeta reflects dynamic integration of a positive signal provided by GnRH from hypothalamic neurons and negative signals emanating from gonadal steroids. Although alphaGSU and LHbeta genes respond transcriptionally in the same manner to changes in hormonal input, different combinations of regulatory elements orchestrate their response. These hormone-responsive regulatory elements are also integral members of much larger combinatorial codes responsible for targeting expression of alphaGSU and LHbeta genes to gonadotropes. In this review, we will profile the genomic landscape of the promoter-regulatory region of both genes, depicting elements and factors that contribute to gonadotrope-specific expression and hormonal regulation. Within this context, we will highlight the different combinatorial codes that control transcriptional responses, particularly those that mediate the opposing effects of GnRH and one of the sex steroids, androgens. We will use this framework to suggest that GnRH and androgens attain the same transcriptional endpoint through combinatorial codes unique to alphaGSU and LHbeta. This parallelism permits the dynamic and coordinate regulation of two genes that encode a single hormone.

Objective: To describe the development and initial evaluation of the Coping Course, a cognitive-behavioral group intervention designed to enhance general coping and problem-solving skills among incarcerated youth., Method: Between 2001 and 2002, 76 male adolescents incarcerated at a youth correctional facility were assessed by questionnaire and randomly assigned to either the Coping Course (n = 46) or usual care (n = 30). Participants repeated the questionnaire after completion of the intervention. A second correctional facility served as an additional source of control group data (n = 62)., Results: Significant condition x time effects were present for seven of the examined measures: Youth Self-Report externalizing scores, three measures from the Life Attitudes Scale, self-esteem, one measure of social adjustment, and cognitive-behavioral therapy knowledge. Age and race/ethnicity did not moderate effects. Comparing control group participants with youth at a separate correctional facility who did not receive the Coping Course indicated that change was uniquely associated with participation in the intervention., Conclusions: Our goal was to take an efficacious adolescent depression group intervention and modify it for use with youth in correctional facilities. Preliminary findings are promising and provide evidence for future research of cognitive-behavioral group treatments with incarcerated youth.

Objective: To evaluate effectiveness of the Adolescent Coping With Depression (CWD-A) course, a cognitive-behavioral group intervention for depressed adolescents with comorbid conduct disorder., Method: Between 1998 and 2001, 93 nonincarcerated adolescents (ages 13-17 years) meeting criteria for major depressive disorder and conduct disorder were recruited from a county juvenile justice department and randomly assigned to the CWD-A or a life skills/tutoring control condition. Participants were assessed post-treatment and at 6- and 12-month follow-up. Dichotomous outcomes were analyzed with logistic regression; dimensional measures were analyzed using random effects regression., Results: Major depressive disorder recovery rates post-treatment were greater in CWD-A (39%) compared with life skills/tutoring control (19%) (odds ratio 2.66, 95% confidence interval = 1.03-6.85). CWD-A participants reported greater reductions in Beck Depression Inventory-II (r2 = 0.055, p =.033) and Hamilton Depression Rating Scale (r2 = 0.047, p =.039) scores and improved social functioning (r2 = 0.064, p =.019) post-treatment. Group differences in major depressive disorder recovery rates at 6- and 12-month follow-up were nonsignificant, as were differences in conduct disorder both post-treatment and during follow-up., Conclusions: This is the first randomized, controlled trial of a psychosocial intervention with adolescents with major depressive disorder and conduct disorder. Although the CWD-A appears to be an effective acute treatment for depression in adolescents with multiple disorders, findings emphasize the need to improve long-term outcomes for depressed adolescents with psychiatric comorbidity and imply that interventions for comorbid populations focus directly on each specific disorder.

In the prostate, androgens negatively regulate the expression of transforming growth factor-beta (TGF-beta) ligands and receptors and Smad activation through unknown mechanisms. We show that androgens (dihydrotestosterone and R1881) down-regulate TGF-beta1-induced expression of TGF-beta1, c-Fos, and Egr-1 in the human prostate adenocarcinoma cell line, LNCaP. Moreover, 5alpha-dihydrotestosterone (DHT) inhibits TGF-beta1 activation of three TGF-beta1-responsive promoter constructs, 3TP-luciferase, AP-1-luciferase, and SBE4(BV)-luciferase, in LNCaP cells either with or without enforced expression of TGF-beta receptors (TbetaRI and TbetaRII). Similarly, DHT inhibits the activation of Smad-binding element (SBE)4(BV)-luciferase by either constitutively activated TbetaRI (T204D) or constitutively activated Smad3 (S3*). Activation of SBE4(BV)-luciferase by S3* in the NRP-154 prostatic cell line, which is androgen receptor (AR)-negative but highly responsive to TGF-beta1, is blocked by co-transfection with either full-length AR or AR missing the DNA binding domain. Immunoprecipitation and GST pull-down assays show that AR directly associates with Smad3 but not Smad2 or Smad4. Electrophoretic mobility shift assays indicate that the AR ligand binding domain directly inhibits the association of Smad3 to the Smad-binding element. In conclusion, our data demonstrate for the first time that ligand-bound AR inhibits TGF-beta transcriptional responses through selectively repressing the binding of Smad3 to SBE.

Synthesis of LH is suppressed by feedback from gonadal steroids. Previously, we demonstrated that 779 bp of the bovine LHbeta promoter was sufficient to target expression of a chloramphenicol acetyltransferase reporter gene specifically to the pituitary in transgenic mice, and found that it was appropriately suppressed after administration of T or E2. In this study, we report that ligand-bound AR, but not ligand-bound ER, directly suppressed activity of the bovine LHbeta promoter when examined in a gonadotrope-derived cell line. Additional studies with mutated bovine LHbeta promoter constructs focused on the proximal 5'-flanking region because of the presence of several cis-acting elements that are highly conserved across all mammals. These include regulatory elements that bind steroidogenic factor 1 (SF-1), Egr-1, and Pitx1. When tested by cotransfection with AR, overexpression of Egr-1, Pitx1, and constitutively active steroidogenic factor 1 (SF-1DeltaLBD) each individually rescued androgen-mediated suppression of the bovine LHbeta promoter. This suggested a functional interaction between each of these transcription proteins and AR. In contrast, overexpression of full-length SF-1 was incapable of relieving the bovine LHbeta promoter from the suppressive effect imposed by AR. This suggested that the ligand-binding domain of SF-1 plays an important role in functional interactions that occur between this protein and AR. This notion was further supported by binding assays performed with glutathione-S-transferase-AR: these identified SF-1 as a key interactive partner and localized this interaction to the ligand-binding domain of the protein. Additional binding studies indicated that protein interactions between SF-1, Pitx1, and Egr-1 interfere with formation of a binary complex that contains AR and SF-1. Thus, we conclude that AR suppresses activity of the bovine LHbeta promoter through protein-protein interactions with SF-1 and that the degree of this interaction can be modified by the presence of Egr-1 and Pitx1.

Previously, we reported that the AR directly suppressed transcription of the alpha glycoprotein hormone subunit (alphaGSU) gene in a ligand-dependent fashion while ER had no effect. Mutagenesis studies of the alphaGSU promoter indicated that two elements were required for AR-mediated suppression: the alpha basal element and tandem cAMP response elements (CREs). Because several members of the bZip family of transcriptional proteins can bind the CREs, we used several functional assays to determine whether AR interacts selectively with cJun, activation transcription factor 2 (ATF2), or CRE binding protein (CREB). When tested by cotransfection with AR, cJun and ATF2 specifically rescued androgen-mediated suppression of the alphaGSU-reporter construct in a gonadotrope-derived cell line. In contrast, cotransfected CREB displayed no activity in this rescue assay. In fact, overexpression of CREB alone diminished activity of the alphaGSU promoter, suggesting that the transcriptional activity normally conferred by the tandem CREs in gonadotropes requires their occupancy by cJun/ATF2 heterodimers. Binding assays carried out with a glutathione-S-transferase-AR fusion protein indicated that the receptor itself also displayed a clear preference for binding cJun and ATF2. Furthermore, we ruled out the possibility that AR suppressed activity of the alphaGSU promoter by reducing synthesis of these bZip proteins. Additional experiments suggested that phosphorylation of AR or histone acetylation are unlikely requirements for AR suppression of alphaGSU promoter activity. Thus, our data suggest that AR suppresses activity of the alphaGSU promoter through direct protein-protein interactions with cJun and ATF2.

Due to the complex nature of the anatomy of the equine head, superimposition of numerous structures, and poor soft tissue differentiation, radiography may be of limited value in the diagnosis of basilar skull fractures. However, in many horses radiographic changes such as soft tissue opacification of the guttural pouch region, irregular bone margination at the sphenooccipital line, attenuation of the nasopharynx, ventral displacement of the dorsal pharyngeal wall and the presence of irregularly shaped bone fragments in the region of the guttural pouches are suggestive of a fracture of the skull base. These findings in conjunction with physical examination findings and historical information may lead to a presumptive diagnosis of a fracture. When available and when the patient will accommodate the equipment, computed tomography may give a definitive diagnosis owing to its superior resolution and differentiation of soft tissue structures.

An 8-year-old Thoroughbred gelding was admitted for evaluation of chronic lameness of the left scapulohumeral joint of 3 months' duration. Radiography revealed a radiolucent lesion with the proximal portion of the humerus in the area of the metaphysis. Scintigraphy confirmed radiographic findings, with an increased uptake of technetium Tc 99m medronate in the proximal portion of the left humerus. A preliminary diagnosis of humeral fracture was made. Two weeks later, the horse was readmitted for clinical signs of respiratory distress. Radiographic and ultrasonographic evaluation revealed masses within the thoracic and abdominal cavities. The diagnosis was changed to neoplasm with multiple metastases. Because of the unfavorable prognosis, the horse was euthanatized. Necropsy findings confirmed an aggressive neoplasm. Special histochemical stains, immunohistochemistry, and electron microscopy were required to characterize the neoplasm as an anaplastic fibrosarcoma. Findings in this horse illustrate the importance of considering neoplasia, resulting in bone lesions, as a possible cause of chronic lameness in horses.

This article presents physiologic and pathophysiologic processes that may affect decisions regarding anesthetic protocol. Various organ systems are discussed with specific attention given to pathophysiologic alterations and their effects on anesthesia.