Abstract Background Mammalian reproductive outcomes are influenced by whole-body energy status, especially in high-prolific sows. During lactation, high energy demands often lead to a severe negative energy balance (NEB), which compromises subsequent reproductive performance. The molecular processes underlying the sustained impact of lactational energy insufficiency on post-weaning follicular development are not well understood. We hypothesize that this energy shortage disrupts the development of mural granulosa cells (GCs), ultimately compromising oocyte developmental competence. We aimed to delineate the molecular processes by which energy status regulates mural GC development employing a nutritional intervention. Results Over the 14 days of feed restriction, sows receiving a restricted diet (RES; 3.25 kg/d) lost double the amount of body weight and loin muscle depth compared to full-fed sows (FF; diet 6.5 kg/d). Both ovarian weight and average size of the largest healthy follicle were significantly reduced. RNA-seq analysis identified 2,282 differentially expressed genes in RES GCs, of which 1,531 were upregulated and 751 were downregulated. The most enriched pathway was the cell cycle, corroborated by CDK1 immunostaining. RES GCs showed upregulation of key positive cell cycle regulators (e.g., CCNA1/2 , CCNB1–3 , CCNE2 ), while cell cycle inhibitor CDKN1A (P21) was downregulated. In contrast to FF GCs, in RES GCs several members of the IGFBP family, especially IGFBP7 , strongly correlated with most cell cycle-related genes including FOXO1 , CDKN1A , and follicle size exclusively in RES GCs, but not in FF sows. Proliferation markers ( PCNA and MKI67 ) were also upregulated, while differentiation marker CYP19A1 was downregulated. Conclusion Our data suggest that a severe lactational NEB has a lasting impact on mural GCs after weaning, delaying their transition from proliferation to the terminal differentiation. We propose a potential regulatory axis in which IGFBPs, especially IGFBP7 , may link energy metabolism and follicular development through the AKT-FOXO1-CDKN1A-cell cycle regulatory axis. These molecular alterations likely contribute to the compromised oocyte competence observed following energy restriction. Furthermore, our findings identified IGFBP7 as a candidate biomarker linking metabolic status to follicular development.

Leydig cells (LC) are present in the testicular interstitial tissue, and their main function is to produce testosterone (T) for the maintenance of spermatogenesis and extratesticular androgenic and anabolic functions. LC appear in fetal life, and their hormonal products (T and INSL3) direct male fetal masculinization and early development of genital structures. After the quiescent phase between birth and puberty another adult LC population appears and persists until old age. Their T production is the key hormonal stimulus of the male phenotype. We will review here the development, function, regulation, as well as key pathophysiological aspects of the LC.

Significance: Dimeric nicotinamide nucleotide transhydrogenase (NNT) is embedded in the mitochondrial inner membrane and couples the conversion of NADP+/NADH into NADPH/NAD+ to mitochondrial matrix proton influx. NNT was implied in various cancers, but its physiological role and regulation still remain incompletely understood. Recent Advances: NNT function was analyzed by studying: (1) NNT gene mutations in human (adrenal) glucocorticoid deficiency 4 (GCCD4), (2) Nnt gene mutation in C57BL/6J mice, and (3) the effect of NNT knockdown/overexpression in (cancer) cells. In these three models, altered NNT function induced both common and differential aberrations. Critical Issues: Information on NNT protein expression in GCCD4 patients is still scarce. Moreover, NNT expression levels are tissue-specific in humans and mice and the functional consequences of NNT deficiency strongly depend on experimental conditions. In addition, data from intact cells and isolated mitochondria are often unsuited for direct comparison. This prevents a proper understanding of NNT-linked (patho)physiology in GCCD4 patients, C57BL/6J mice, and cancer (cell) models, which complicates translational comparison. Future Directions: Development of mice with conditional NNT deletion, cell-reprogramming-based adrenal (organoid) models harboring specific NNT mutations, and/or NNT-specific chemical inhibitors/activators would be useful. Moreover, live-cell analysis of NNT substrate levels and mitochondrial/cellular functioning with fluorescent reporter molecules might provide novel insights into the conditions under which NNT is active and how this activity links to other metabolic and signaling pathways. This would also allow a better dissection of local signaling and/or compartment-specific (i.e., mitochondrial matrix, cytosol, nucleus) effects of NNT (dys)function in a cellular context. Antioxid. Redox Signal. 41, 927-956.

Young sows mobilise body reserves to support milk production during lactation, resulting in a negative energy balance (NEB). This NEB affects the development of follicles and oocytes that give rise to the next litter. Decreased IGF1 levels due to a NEB are thought to play a role in this process. As this has hardly been studied in multiparous sows, the current study focused on relations between lactation BW loss (%), metabolic hormones, and follicle development in multiparous sows at Day 0 and Day 4 after weaning. A total of 31 sows of parity 4.7 +/- 2.5 were killed at either Day 0 or Day 4 after weaning. Average BW loss during lactation was 3.3 +/- 4.5%, while average backfat loss was 4.1 +/- 0.3 mm. The metabolic profile confirmed the metabolic impact of lactation as both non-esterified fatty acid (NEFA), and creatinine levels were higher at Day 0 than that at Day 4. Conversely, serum levels of IGF1 and growth differentiation factor 15 levels were lower on Day 0 than on Day 4. A higher BW loss (%) was related to higher NEFA levels on Day 0, but not on Day 4. IGF1 concentrations in serum and follicle fluid were similar at Day 0 and Day 4 and were not related to follicle size on these days. In conclusion, although lactation affected postweaning metabolic profiles in these multiparous sows, follicle size was not related to these profiles, probably due to the relatively mild BW loss of these sows. IGF1 concentrations were less affected by lactation and did not seem to limit follicle development, as it does in sows experiencing high weight loss. (c) 2024 The Author(s). Published by Elsevier B.V. on behalf of The Animal Consortium. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Metabolic demands of modern hybrid sows have increased over the years, which increases the chance that sows enter a substantial negative energy balance (NEB) during lactation. This NEB can negatively impact reproductive outcome, which is especially evident in primiparous sows causing a reduced second parity reproductive performance. The negative effects of the lactational NEB on reproductive performance can be partly explained by the influence of the premating metabolic state, during and after lactation, on the development of follicles from which oocytes will give rise to the next litter. In addition, the degree and type of body tissue mobilization during lactation that is, adipose tissue or lean mass, highly influences follicular development. Research investigating relations between the premating metabolic state and follicular and oocyte competence in modern hybrid sows, which experience higher metabolic demands during lactation, is limited. In this review we summarize current knowledge of physiological relations between the metabolic state of modern hybrid sows and follicular developmental competence. In addition, we discuss potential implications of these relations for current sow management strategies.

AbstractMicroplastics have been documented in drinking water, but their effects on human health from ingestion, or the concentrations at which those effects begin to manifest, are not established. Here, we report on the outcome of a virtual expert workshop conducted between October 2020 and October 2021 in which a comprehensive review of mammalian hazard studies was conducted. A key objective of this assessment was to evaluate the feasibility and confidence in deriving a human health-based threshold value to inform development of the State of California’s monitoring and management strategy for microplastics in drinking water. A tiered approach was adopted to evaluate the quality and reliability of studies identified from a review of the peer-reviewed scientific literature. A total of 41 in vitro and 31 in vivo studies using mammals were identified and subjected to a Tier 1 screening and prioritization exercise, which was based on an evaluation of how each of the studies addressed various quality criteria. Prioritized studies were identified largely based on their application and reporting of dose–response relationships. Given that methods for extrapolating between in vitro and in vivo systems are currently lacking, only oral exposure in vivo studies were identified as fit-for-purpose within the context of this workshop. Twelve mammalian toxicity studies were prioritized and subjected to a Tier 2 qualitative evaluation by external experts. Of the 12 studies, 7 report adverse effects on male and female reproductive systems, while 5 reported effects on various other physiological endpoints. It is notable that the majority of studies (83%) subjected to Tier 2 evaluation report results from exposure to a single polymer type (polystyrene spheres), representing a size range of 0.040 to 20 µm. No single study met all desired quality criteria, but collectively toxicological effects with respect to biomarkers of inflammation and oxidative stress represented a consistent trend. While it was possible to derive a conservative screening level to inform monitoring activities, it was not possible to extrapolate a human–health-based threshold value for microplastics, which is largely due to concerns regarding the relative quality and reliability of current data, but also due to the inability to extrapolate data from studies using monodisperse plastic particles, such as polystyrene spheres to an environmentally relevant exposure of microplastics. Nevertheless, a conservative screening level value was used to estimate a volume of drinking water (1000 L) that could be used to support monitoring activities and improve our overall understanding of exposure in California’s drinking water. In order to increase confidence in our ability to derive a human–health-based threshold value in the future, several research recommendations are provided, with an emphasis towards strengthening how toxicity studies should be conducted in the future and an improved understanding of human exposure to microplastics, insights critically important to better inform future risk assessments. Graphical abstract

Bisphenol A (BPA) is an environmental endocrine disruptor. Recent studies have shown an association between decreased spermatogenesis and gut microbiota alteration. However, the potential associations and mechanisms of BPA exposure on spermatogenesis, hormone production, and gut microbiota remain unknown. This study aims to investigate BPA-induced male reproductive toxicity and the potential link with gut microbiota dysbiosis. Male Sprague Dawley rats were exposed to BPA at different doses by oral gavage for thirty consecutive days. The extent of testicular damage was evaluated by basic parameters of body weight and hematoxylin-eosin (H&E) staining. Next, we determined the mRNA levels and protein levels of apoptosis, histone-related factors, and mammalian target of rapamycin (mTOR) pathway in testes. Finally, 16 S rDNA sequencing was used to analyze gut microbiota composition after BPA exposure. BPA exposure damaged testicular histology, significantly decreased sperm count, and increased sperm abnormalities. In addition, BPA exposure caused oxidative stress and cell apoptosis in testes. The levels of histone (H2A, H3) were significantly increased, while ubiquitin histone H2A (ub-H2A) and ubiquitin histone H2B (ub-H2B) were markedly reduced. Furthermore, BPA activated the PI3K and AKT expression, but the protein expressions of mTOR and 4EBP1 in testes were inhibited significantly. Additionally, the relative abundance of class Gammaproteobacteria, and order Betaproteobacteriales was significantly higher when treated with a high dose of BPA compared to the control group, which was negatively correlated with testosterone level. This study highlights the relationship between BPA-induced reproductive toxicity and gut microbiota disorder and provides new insights into the prevention and treatment of BPA-induced reproductive damage.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Long non-coding RNAs (lncRNAs) play important roles in multiple biological processes including ovarian follicular development. Here we aimed to gain novel information regarding lncRNAs transcriptome profiles in porcine granulosa cells of advanced atretic antral (AA) and healthy antral (HA) follicles using RNA-seq. A total of 11,321 lncRNAs including 10,813 novel and 508 annotated lncRNAs were identified, of which 173 lncRNAs were differentially expressed (DE-lncRNAs); ten of these were confirmed by qRT-PCR. Gene Ontology indicated that DE-lncRNAs associated with developmental processes were highly enriched. Pathway analysis demonstrated predicted cis- and trans-targets of DE-lncRNAs. Potential mRNA targets of up-regulated DE-lncRNAs were mainly enriched in apoptosis related pathways, while targeted genes of downregulated DE-lncRNAs were primarily enriched in metabolism and ovarian steroidogenesis pathways. Linear regression analyses showed that expression of upregulated DE-lncRNAs was significantly associated with apoptosis related genes. NOVEL_00001850 is the most-downregulated DE-lncRNA (FDR = 0.04, FC = −6.53), of which miRNA binding sites were predicted. KEGG analysis of its downregulated target genes revealed that ovarian steroidogenesis was the second most highlighted pathway. qRT-PCR and linear regression analysis confirmed the expression and correlation of its potential targeted gene, CYP19A1, a key gene involved in estradiol synthesis. Our results indicate that lncRNAs may participate in granulosa cells apoptosis and thus antral follicular atresia.

One of the main causes of female infertility is a deregulated antral follicular atresia, a process of which the underlying molecular mechanisms are largely unknown. Our objective was therefore to characterize the complex transcriptome changes in porcine granulosa cells of healthy antral (HA) and advanced antral atretic (AA) follicles, using ELISA and RNA-Seq followed by qRT-PCR and immunohistochemistry. Granulosa cell RNA-Seq data revealed 2160 differentially expressed genes, 1483 with higher and 677 with lower mRNA concentrations in AA follicles. Bioinformatic analysis showed that the upregulated genes in AA follicles were highly enriched in inflammation and apoptosis processes, while the downregulated transcripts were mainly highlighted in the steroid biosynthesis pathway and response to oxidative stress processes including antioxidant genes (e.g., GSTA1, GCLC, GCLM, IDH1, GPX8) involved in the glutathione metabolism pathway and other redox-related genes (e.g., RRM2B, NDUFS4). These observations were confirmed by RT-qPCR and immunohistochemistry. Additionally, the granulosa cells of AA follicles express significantly stronger 8-OHdG immunostaining, a marker of oxidative DNA damage, implicating that oxidative stress may participate in follicular atresia. We hypothesize that the decrease in anti-apoptotic factors and steroid hormones coincides with increased oxidative stress markers and the expression of pro-apoptotic factors, all contributing to antral follicular atresia.

EDITORIAL article Front. Cell Dev. Biol., 24 June 2021Sec. Cell Growth and Division Volume 9 - 2021 | https://doi.org/10.3389/fcell.2021.712467

In the present investigation, we examined whether a change in whole body energy fluxes could affect ovarian follicular development, employing mice ectopically expressing uncoupling protein 1 in skeletal muscle (UCP1-TG). Female UCP1-TG and wild-type (WT) mice were dissected at the age of 12 weeks. Energy intake and expenditure, activity, body weight and length, and body composition were measured. Plasma insulin, glucose, leptin, plasma fibroblast growth factor 21 (FGF21) and plasma insulin-like growth factor 1 (IGF1) levels were analyzed and ovarian follicle and corpus luteum numbers were counted. IGF1 signaling was analyzed by immunohistochemical staining for the activation of insulin receptor substrate 1/2 (IRS1/2) and AKT. UCP1-TG female mice had increased energy expenditure, reduced body size, maintained adiposity, and decreased IGF1 concentrations compared to their WT littermates, while preantral and antral follicle numbers were reduced by 40% and 60%, respectively. Corpora lutea were absent in 40% of the ovaries of UCP1-TG mice. Phospho-IRS1, phospho-AKT -Ser473 and -Thr308 immunostaining was present in the granulosa cells of antral follicles in WT ovaries, but faint to absent in the antral follicles of UCP1-TG mice. In conclusion, the reduction in circulating IGF1 levels due to the ectopic expression of UCP1 is associated with reduced immunostaining of the IRS1-PI3/AKT pathway, which may negatively affect ovarian follicle development and ovulation.

BACKGROUND:Origin of human adult Leydig cells (ALCs) is not well understood. This might be partly due to limited data available on the identification and location of human precursor and stem Leydig cells (SLCs) which hampers the study on the development of ALCs.OBJECTIVES:The aim of the present study was to investigate whether described human (PDGFRα, NGFR) and rodent (NES, PDGFRα, THY1, NR2F2) SLC markers are expressed by a common cell population within human adult testicular interstitial cells in vivo and before and after in vitro propagation.MATERIALS AND METHODS:Immunohistochemical analyses were used to identify localization of human adult testicular interstitial cells expressing described SLC markers. Next, interstitial cells were isolated and cultured. The percentage of cells expressing one or more SLC markers was determined before and after culture using flow cytometry.RESULTS:NR2F2 and PDGFRα were present in peritubular, perivascular, and Leydig cells, while THY1 was expressed in peritubular and perivascular cells. Although NES and NGFR were expressed in endothelial cells, co-localization with PDGFRα was found for both in vitro, although for NGFR only after culture. All marker positive cells were able to undergo propagation in vitro.DISCUSSION:The partly overlap in localization and overlap in expression in human testicular cells indicate that PDGFRα, NR2F2, and THY1 are expressed within the same ALC developmental lineage from SLCs. Based on the in vitro results, this is also true for NES and after in vitro propagation for NGFR.CONCLUSION:Our results that earlier described SLC markers are expressed in overlapping human interstitial cell population opens up further research strategies aiming for a better insight in the Leydig cell lineage and will be helpful for development of strategies to cure ALC dysfunction.

Identification of reliable characteristics of follicle quality and developmental competence has been pursued in numerous studies, but with inconsistent outcomes. Here, we aimed to identify these characteristics by analysis of the follicular fluid (FF) steroid profile in relation to cumulus-oocyte complex (COC) morphology and follicle size, followed by molecular substantiation. Multiparous sows at weaning were used to facilitate analysis at the start of the follicular phase of the oestrus cycle. Sows with a higher average follicle size (>= 5 mm vs. <5 mm) had a higher follicular fluid beta-estradiol concentration, but did not differ in other measured steroids. Sows with high compared to low percentage high-quality COCs (<70% vs. >= 70% high-quality) had follicular fluid with a higher concentration of beta-estradiol, 19-norandrostenedione, progesterone, and alpha-testosterone, while the concentration of cortisol was lower. Transcriptome analysis of granulosa cells of healthy follicles of sows with a high percentage high-quality COCs showed higher abundance of transcripts involved in ovarian steroidogenesis (e.g., CYP19A2 and 3, POR, VEGFA) and growth (IGF1) and differential abundance of transcripts involved in granulosa cell apoptosis (e.g., GADD45A, INHBB). Differences in aromatase transcript abundance (CYP19A1, 2 and 3) were confirmed at the protein level. In addition, sows with a high percentage high-quality COCs lost less weight during lactation and had higher plasma IGF1 concentration at weaning, which may have affected COC quality. To the best of our knowledge, this study is also the first to report the relation between FF steroid profile and COC quality.Sows with a higher percentage high-quality COCs have a distinct follicular fluid steroid profile and concomitant granulosa cell transcriptome.

Selection for prolificacy in sows has resulted in higher metabolic demands during lactation. In addition, modern sows have an increased genetic merit for leanness. Consequently, sow metabolism during lactation has changed, possibly affecting milk production and litter weight gain. The aim of this study was to investigate the effect of lactational feed intake on milk production and relations between mobilization of body tissues (adipose tissue or skeletal muscle) and milk production in modern sows with a different lactational feed intake. A total of 36 primiparous sows were used, which were either full-fed (6.5 kg/day) or restricted-fed (3.25 kg/day) during the last 2 weeks of a 24-day lactation. Restricted-fed sows had a lower milk fat percentage at weaning and a lower litter weight gain and estimated milk fat and protein production in the last week of lactation. Next, several relations between sow body condition (loss) and milk production variables were identified. Sow BW, loin muscle depth and backfat depth at parturition were positively related to milk fat production in the last week of lactation. In addition, milk fat production was related to the backfat depth loss while milk protein production was related to the loin muscle depth loss during lactation. Backfat depth and loin muscle depth at parturition were positively related to lactational backfat depth loss or muscle depth loss, respectively. Together, results suggest that sows which have more available resources during lactation, either from a higher amount of body tissues at parturition or from an increased feed intake during lactation, direct more energy toward milk production to support a higher litter weight gain. In addition, results show that the type of milk nutrients that sows produce (i.e. milk fat or milk protein) is highly related to the type of body tissues that are mobilized during lactation. Interestingly, relations between sow body condition and milk production were all independent of feed level during lactation. Sow management strategies to increase milk production and litter growth in modern sows may focus on improving sow body condition at the start of lactation or increasing feed intake during lactation.

Circular RNAs (circRNAs) are thought to play essential roles in multiple biological processes, including apoptosis, an important process in antral follicle atresia. We aimed to investigate the potential involvement of circRNAs in granulosa cell apoptosis and thus antral follicle atresia. CircRNA expression profiles were generated from porcine granulosa cells isolated from healthy antral (HA) and atretic antral (AA) follicles. Over 9632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). Back-splicing, RNase R resistance, and stability of DE-circRNAs were validated, and miRNA binding sites and related target genes were predicted. Two exonic circRNAs with low false discovery rate (FDR) high fold change, miRNA binding sites, and relevant biological functions—circ_CBFA2T2 and circ_KIF16B—were selected for further characterization. qRT-PCR and linear regression analysis confirmed expression and correlation of the targeted genes—the antioxidant gene GCLC (potential target of circ_CBFA2T2) and the apoptotic gene TP53 (potential target of circ_KIF16B). Increased mRNA content of TP53 in granulosa cells of AA follicles was further confirmed by strong immunostaining of both p53 and its downstream target pleckstrin homology like domain family a member 3 (PHLDA3) in AA follicles compared to negligible staining in granulosa cells of HA follicles. Therefore, we concluded that aberrantly expressed circRNAs presumably play a potential role in antral follicular atresia.

Antral follicle size might be a valuable additive predictive marker for IVF outcome. To better understand consequences of antral follicle size as a marker for reproductive outcome, we aimed to obtain insight in follicle size-related granulosa cell processes, as granulosa cells play an essential role in follicular development via the production of growth factors, steroids and metabolic intermediates. Using the pig as a model, we compared gene expression in granulosa cells of smaller and larger follicles in the healthy antral follicle pool of sows, which had a high variation versus low variation in follicle size. Selected gene expression was confirmed at the protein level. Granulosa cells of smaller antral follicles showed increased cell proliferation, which was accompanied by a metabolic shift towards aerobic glycolysis (i.e. the Warburg effect), similar to other highly proliferating cells. High granulosa cell proliferation rates in smaller follicles might be regulated via increased granulosa cell expression of the androgen receptor and the epidermal growth factor receptor, which are activated in response to locally produced mitogens. While granulosa cells of smaller follicles in the pool are more proliferative, granulosa cells of larger follicles express more maturation markers such as insulin-like growth factor-1 (IGF1) and angiopoietin 1 (ANGPT1) and are therefore more differentiated. As both higher IGF1 and ANGPT1 have been associated with better IVF outcomes, the results of our study imply that including smaller follicles for oocyte aspiration might have negative consequences for IVF outcome.

Abstract STUDY QUESTION Is it possible to differentiate primary human testicular platelet-derived growth factor receptor alpha positive (PDGFRα+) cells into functional Leydig cells? SUMMARY ANSWER Although human testicular PDGFRα+ cells are multipotent and are capable of differentiating into steroidogenic cells with Leydig cell characteristics, they are not able to produce testosterone after differentiation. WHAT IS KNOWN ALREADY In rodents, stem Leydig cells (SLCs) that have been identified and isolated using the marker PDGFRα can give rise to adult testosterone-producing Leydig cells after appropriate differentiation in vitro. Although PDGFRα+ cells have also been identified in human testicular tissue, so far there is no evidence that these cells are true human SLCs that can differentiate into functional Leydig cells in vitro or in vivo. STUDY DESIGN, SIZE, DURATION We isolated testicular cells enriched for interstitial cells from frozen–thawed fragments of testicular tissue from four human donors. Depending on the obtained cell number, PDGFRα+-sorted cells of three to four donors were exposed to differentiation conditions in vitro to stimulate development into adipocytes, osteocytes, chondrocytes or into Leydig cells. We compared their cell characteristics with cells directly after sorting and cells in propagation conditions. To investigate their differentiation potential in vivo, PDGFRα+-sorted cells were transplanted in the testis of 12 luteinizing hormone receptor-knockout (LuRKO) mice of which 6 mice received immunosuppression treatment. An additional six mice did not receive cell transplantation and were used as a control. PARTICIPANTS/MATERIALS, SETTING, METHODS Human testicular interstitial cells were cultured to Passage 3 and FACS sorted for HLA-A,B,C+/CD34−/PDGFRα+. We examined their mesenchymal stromal cell (MSC) membrane protein expression by FACS analyses. Furthermore, we investigated lineage-specific staining and gene expression after MSC trilineage differentiation. For the differentiation into Leydig cells, PDGFRα+-sorted cells were cultured in either proliferation or differentiation medium for 28 days, after which they were stimulated either with or without hCG, forskolin or dbcAMP for 24 h to examine the increase in gene expression of steroidogenic enzymes using qPCR. In addition, testosterone, androstenedione and progesterone levels were measured in the culture medium. We also transplanted human PDGFRα+-sorted testicular interstitial cells into the testis of LuRKO mice. Serum was collected at several time points after transplantation, and testosterone was measured. Twenty weeks after transplantation testes were collected for histological examination. MAIN RESULTS AND THE ROLE OF CHANCE From primary cultured human testicular interstitial cells at Passage 3, we could obtain a population of HLA-A,B,C+/CD34−/PDGFRα+ cells by FACS. The sorted cells showed characteristics of MSC and were able to differentiate into adipocytes, chondrocytes and osteocytes. Upon directed differentiation into Leydig cells in vitro, we observed a significant increase in the expression of HSD3B2 and INSL3. After 24 h stimulation with forskolin or dbcAMP, a significantly increased expression of STAR and CYP11A1 was observed. The cells already expressed HSD17B3 and CYP17A1 before differentiation but the expression of these genes were not significantly increased after differentiation and stimulation. Testosterone levels could not be detected in the medium in any of the stimulation conditions, but after stimulation with forskolin or dbcAMP, androstenedione and progesterone were detected in culture medium. After transplantation of the human cells into the testes of LuRKO mice, no significant increase in serum testosterone levels was found compared to the controls. Also, no human cells were identified in the interstitium of mice testes 20 weeks after transplantation. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION This study was performed using tissue from only four donors because of limitations in donor material. Because of the need of sufficient cell numbers, we first propagated cells to passage 3 before FACS of the desired cell population was performed. We cannot rule out this propagation of the cells resulted in loss of stem cell properties. WIDER IMPLICATIONS OF THE FINDINGS A lot of information on Leydig cell development is obtained from rodent studies, while the knowledge on human Leydig cell development is very limited. Our study shows that human testicular interstitial PDGFRα+ cells have different characteristics compared to rodent testicular PDGFRα+ cells in gene expression levels of steroidogenic enzymes and potential to differentiate in adult Leydig cells under comparable culture conditions. This emphasizes the need for confirming results from rodent studies in the human situation to be able to translate this knowledge to the human conditions, to eventually contribute to improvements of testosterone replacement therapies or establishing alternative cell therapies in the future, potentially based on SLCs. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Amsterdam UMC, location AMC, Amsterdam, the Netherlands. All authors declare no competing interests.