{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:34:15Z","timestamp":1787027655086,"version":"build-2736575974"},"reference-count":62,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,27]],"date-time":"2025-12-27T00:00:00Z","timestamp":1766793600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Koala Research Foundation Australia Ltd"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Ovarian interstitial tissue (IT) is a prominent but poorly characterised component of the koala ovary. This study analysed the morphology and immunohistochemical profile of IT in the koala ovary across different reproductive phases. Ovaries from ten sexually mature females were examined histologically and immunolabelled for aromatase, HSD3B2, HSD17B1, the follicle stimulating hormone receptor (FSHR), and the luteinizing hormone receptor (LHR). IT occurred as multifocal cortical aggregates composed of two distinct cell types. Large interstitial cells were polygonal with highly vacuolated and weakly eosinophilic cytoplasm and were morphologically consistent with a steroidogenic phenotype. Small interstitial cells were densely eosinophilic, non-vacuolated, and morphologically uniform across reproductive phases. Immunohistochemical analysis revealed that small interstitial cells exhibited the broadest expression of steroidogenic markers in the ovary, with coexpression of HSD3B2, HSD17B1, aromatase, FSHR, and LHR. In contrast, large interstitial cells exhibited comparatively limited enzyme and receptor expression. Staining intensity across all markers was strongest during the interoestrous phase, moderate in the proliferative phase, reduced in the luteal phase, and minimal or absent when the koalas were lactating, suggesting potential cyclical variation in IT function. Granulosa, theca and germinal epithelial cells showed variable expression of steroidogenic enzymes and gonadotropin receptors, indicating that ovarian steroidogenesis in the koala may be more heterogeneous than predicted by the classical two-cell, two-gonadotropin model. Together, these findings provide preliminary evidence for the steroidogenic capacity of ovarian IT in the koala, indicating that its activity varies across reproductive phases, and appear to suggest a unique cellular organisation compared to other mammals. As these results are based on morphology and immunohistochemistry alone, further functional studies are required to confirm the steroidogenic output and clarify the physiological significance of IT in this species.<\/jats:p>","DOI":"10.3390\/biology15010047","type":"journal-article","created":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T06:25:51Z","timestamp":1766989551000},"page":"47","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Steroidogenic Capacity of Ovarian Interstitial Tissue in the Koala (Phascolarctos cinereus): Morphological and Immunohistochemical Evidence"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-7249-0264","authenticated-orcid":false,"given":"Jackson","family":"Boyd","sequence":"first","affiliation":[{"name":"School of Environment, The University of Queensland, Gatton 4343, Australia"},{"name":"School of Veterinary Science, The University of Queensland, Gatton 4343, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0290-5458","authenticated-orcid":false,"given":"Stephen D.","family":"Johnston","sequence":"additional","affiliation":[{"name":"School of Environment, The University of Queensland, Gatton 4343, Australia"},{"name":"School of Veterinary Science, The University of Queensland, Gatton 4343, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5791-6066","authenticated-orcid":false,"given":"Chiara","family":"Palmieri","sequence":"additional","affiliation":[{"name":"School of Veterinary Science, The University of Queensland, Gatton 4343, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1966","DOI":"10.1111\/1365-2664.13127","article-title":"Management of multiple threats achieves meaningful koala conservation outcomes","volume":"55","author":"Beyer","year":"2018","journal-title":"J. Appl. Ecol."},{"key":"ref_2","unstructured":"Department of Environment and Science (2023, April 16). Koala Conservation, Available online: https:\/\/environment.des.qld.gov.au\/wildlife\/animals\/living-with\/koalas\/facts."},{"key":"ref_3","unstructured":"Comizzoli, P., Brown, J.L., and Holt, W.V. (2014). The Koala (Phascolarctos cinereus): A Case Study in the Development of Reproductive Technology in a Marsupial. Reproductive Sciences in Animal Conservation, Springer."},{"key":"ref_4","unstructured":"Comizzoli, P., Brown, J.L., and Holt, W.V. (2019). Using the Koala (Phascolarctos cinereus) as a Case Study to Illustrate the Development of Artificial Breeding Technology in Marsupials: An Update. Reproductive Sciences in Animal Conservation, Springer International Publishing. [2nd ed.]."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Pagliarani, S., Palmieri, C., McGowan, M., Carrick, F., Boyd, J., and Johnston, S.D. (2023). Anatomy of the Female Koala Reproductive Tract. Biology, 12.","DOI":"10.3390\/biology12111445"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1530\/rep.1.00300","article-title":"Semen-induced luteal phase and identification of a LH surge in the koala (Phascolarctos cinereus)","volume":"128","author":"Johnston","year":"2004","journal-title":"Reproduction"},{"key":"ref_7","first-page":"433","article-title":"On the Corpora Lutea and Interstitial Tissue of the Ovary in the Marsupialia","volume":"61","year":"1916","journal-title":"J. Cell Sci."},{"key":"ref_8","unstructured":"Johnston, S.D. (1999). Studies Towards the Development of an Artificial Insemination Protocol in the Koala, Phascolarctos cinereus. [Ph.D. Thesis, The University of Queensland]."},{"key":"ref_9","unstructured":"Pagliarani, S. (2021). Further Insights into the Pathogenesis of Chlamydiosis and Characterisation of Immune Cells in the Reproductive Tract of Koalas (Phascolarctos cinereus). [Ph.D. Thesis, The University of Queensland]."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1002\/ar.1091490311","article-title":"A Comparative Histochemical Study of Interstitial Tissue and Follicular Atresia in the Mammalian Ovary","volume":"149","author":"Guraya","year":"1964","journal-title":"Anat. Rec."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/S0074-7696(08)61889-6","article-title":"Recent advances in the morphology, histochemistry, biochemistry, and physiology of interstitial gland cells of mammalian ovary","volume":"55","author":"Guraya","year":"1978","journal-title":"Int. Rev. Cytol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1006\/gcen.1999.7331","article-title":"Studies on the origin of ovarian interstitial tissue and the incidence of endometrial hyperplasia in domestic and feral cats","volume":"116","author":"Perez","year":"1999","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_13","first-page":"251","article-title":"Origins and development of interstitial tissue in ovaries of rabbit and guinea-pig","volume":"113","author":"Deanesly","year":"1972","journal-title":"J. Anat."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"872","DOI":"10.1111\/rda.13178","article-title":"Histological and immunohistochemical investigations of ovarian interstitial glands during non-breeding season in camels (Camelus dromedarius)","volume":"53","author":"Awad","year":"2018","journal-title":"Reprod. Domest. Anim."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1002\/aja.1001150204","article-title":"Cyclic changes of interstitial gland tissue of the human ovary","volume":"115","author":"Mossman","year":"1964","journal-title":"Am. J. Anat."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1016\/S0303-7207(02)00058-8","article-title":"The corpus luteum and interstitial tissue in a marsupial, the brushtail possum (Trichosurus vulpecula)","volume":"191","author":"Eckery","year":"2002","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1095\/biolreprod.103.015446","article-title":"Determination of steroidogenic potential of ovarian cells of the brushtail possum (Trichosurus vulpecula)","volume":"69","author":"Whale","year":"2003","journal-title":"Biol. Reprod."},{"key":"ref_18","unstructured":"Alcorn, G.T. (1975). Development of the Ovary and Urino-Genital Ducts in the Tammar Wallaby Macropus eugenii (Desmarest, 1817). [Ph.D. Thesis, Macquarie University]."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"901","DOI":"10.1210\/endo-80-5-901","article-title":"A Functional Role for 20a-Hydroxypregn-4-en-3-one in the Rabbit","volume":"80","author":"Hilliard","year":"1967","journal-title":"Endocrinology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1457","DOI":"10.1071\/RD19034","article-title":"Ovary of the southern hairy-nosed wombat (Lasiorhinus latifrons): Its divergent structural organisation","volume":"31","author":"Breed","year":"2019","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1530\/jrf.0.1060207","article-title":"High androgen production by ovarian thecal interstitial cells: A mechanism for delayed ovulation in a tropical vespertilionid bat, Scotophilus heathi","volume":"106","author":"Abhilasha","year":"1996","journal-title":"J. Reprod. Fertil."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/j.jsbmb.2009.11.010","article-title":"Sequence\u2013function correlation of aromatase and its interaction with reductase","volume":"118","author":"Hong","year":"2010","journal-title":"J. Steroid Biochem. Mol. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.mce.2003.11.018","article-title":"Structure\/function aspects of human 3\u03b2-hydroxysteroid dehydrogenase","volume":"215","author":"Thomas","year":"2004","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1016\/j.gene.2016.04.031","article-title":"Current knowledge of the multifunctional 17\u03b2-hydroxysteroid dehydrogenase type 1 (HSD17B1)","volume":"588","author":"He","year":"2016","journal-title":"Gene"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1903","DOI":"10.2527\/1998.7671903x","article-title":"Expression of steroidogenic enzyme and gonadotropin receptor genes in bovine follicles during ovarian follicular waves: A review","volume":"76","author":"Bao","year":"1998","journal-title":"J. Anim. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1530\/reprod\/120.1.49","article-title":"Studies of the oestrous cycle, oestrus and pregnancy in the koala (Phascolarctos cinereus)","volume":"120","author":"Johnston","year":"2000","journal-title":"Reproduction"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1016\/0960-0760(92)90307-5","article-title":"Steroidogenic enzymes: Structure, function, and role in regulation of steroid hormone biosynthesis","volume":"43","author":"Hanukoglu","year":"1992","journal-title":"J. Steroid Biochem. Mol. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Johnston, S.D., Boyd, J., Palacios, P.D., Grosmaire, J., Lee, A., Hulse, L., Vega, L., Pyne, M., Gambini, A., and Palmieri, C. (2025). Evidence of folliculogenesis and the potential of oocyte recovery from koalas with different levels of reproductive pathology. Biology, 14.","DOI":"10.3390\/biology14101435"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1838","DOI":"10.1071\/RD14384","article-title":"Plasma prolactin concentrations during lactation, pouch young development and the return to behavioural oestrus in captive koalas (Phascolarctos cinereus)","volume":"28","author":"Ballentyne","year":"2016","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"829","DOI":"10.1177\/0300985820960132","article-title":"Validating immunohistochemistry assay specificity in investigative studies: Considerations for a weight of evidence approach","volume":"58","author":"Webster","year":"2021","journal-title":"Vet. Pathol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/S0165-2427(99)00115-4","article-title":"Functional and structural comparison of cytokines in different species","volume":"72","author":"Scheerlinck","year":"1999","journal-title":"Vet. Immunol. Immunopathol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1080\/19420862.2015.1100787","article-title":"The European antibody network\u2019s practical guide to finding and validating suitable antibodies for research","volume":"8","author":"Roncador","year":"2016","journal-title":"MAbs"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"106099","DOI":"10.1016\/j.jsbmb.2022.106099","article-title":"The intracellular cholesterol pool in steroidogenic cells plays a role in basal steroidogenesis","volume":"220","author":"Bassi","year":"2022","journal-title":"J. Steroid Biochem. Mol. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1002\/aja.1001230304","article-title":"Studies on the fine structure of ovarian steroid-secreting cells in the rabbit. I. The normal interstitial cells","volume":"123","author":"Davies","year":"1968","journal-title":"Am. J. Anat."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Morrison, O., and Thankur, J. (2021). Molecular complexes at euchromatin, heterochromatin and centromeric chromatin. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22136922"},{"key":"ref_36","first-page":"788","article-title":"Morphological and Endocrine Study of the Ovarian Interstitial Tissue of Viscacha (Lagostomus maximus maximus)","volume":"290","author":"Gil","year":"2007","journal-title":"Reprod. Syst."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1530\/acta.0.072S005","article-title":"Interstitial gland tissue of mammalian ovary","volume":"71","author":"Guraya","year":"1973","journal-title":"Eur. J. Endocrinol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1186\/1477-7827-12-128","article-title":"Human steroidogenesis: Implications for controlled ovarian stimulation with exogenous gonadotropins","volume":"12","author":"Andersen","year":"2014","journal-title":"Reprod. Biol. Endocrinol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Zheng, M., Andersen, C.Y., Rasmussen, F.R., Cadenas, J., Christensen, S.T., and Mamsen, L.S. (2023). Expression of genes and enzymes involved in ovarian steroidogenesis in relation to human follicular development. Front. Endocrinol., 14.","DOI":"10.3389\/fendo.2023.1268248"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1530\/rep.0.1250065","article-title":"Species differences in the ovarian distribution of 3beta-hydroxysteroid dehydrogenase\/delta5-->4 isomerase (3beta-HSD) in two marsupials: The brushtail possum Trichosurus vulpecula and the grey, short-tailed opossum Monodelphis domestica","volume":"125","author":"Ullmann","year":"2003","journal-title":"Reproduction"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/0303-7207(94)90278-X","article-title":"Follicular oestrogen synthesis: The \u2018two-cell, two-gonadotrophin\u2019 model revisited","volume":"100","author":"Hillier","year":"1994","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1007\/s43032-022-00932-z","article-title":"Synthesis, regulatory factors, and signaling pathways of estrogen in the ovary","volume":"30","author":"Xiao","year":"2023","journal-title":"Reprod. Sci."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1210\/jcem-47-2-263","article-title":"17\u03b2-Estradiol Biosynthesis in Cultured Granulosa and Thecal Cells of Human Ovarian Follicles: Stimulation by Follicle-Stimulating Hormone","volume":"47","author":"Moon","year":"1978","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_44","unstructured":"Midgley, A.R., and Salder, W.A. (1979). Regulation of follicular estrogen biosynthesis. Ovarian Follicular Development and Function, Raven Press."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1210\/jcem-26-1-46","article-title":"Steroid Biosynthesis by Human Ovarian Granulosa and Thecal Cells","volume":"26","author":"Ryan","year":"1996","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"590","DOI":"10.1210\/endo-98-3-590","article-title":"Contribution of Granulosa Cells and Follicular Fluid to Ovarian Estrogen Secretion in the Rhesus Monkey in Vivo","volume":"98","author":"Channing","year":"1976","journal-title":"Endocrinology"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1210\/endo-76-1-108","article-title":"Formation of estradiol by granulosa and theca cells ofthe equine ovarian follicle","volume":"76","author":"Ryan","year":"1965","journal-title":"Endocrinology"},{"key":"ref_48","first-page":"143","article-title":"Hormonal and cellular interactions in follicular steroid biosynthesis by the sheep ovary","volume":"30","author":"Armstrong","year":"1981","journal-title":"J. Reprod. Fertil."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1071\/RD99085","article-title":"Immunohistochemical localization of steroidogenic enzymes and comparison with hormone production during follicle development in the pig","volume":"11","author":"Shores","year":"1999","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1006\/gyno.2001.6219","article-title":"Production of steroids by human ovarian surface epithelial cells in culture: Possible role of progesterone as growth inhibitor","volume":"82","author":"Ivarsson","year":"2001","journal-title":"Gynecol. Oncol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1016\/j.tem.2005.07.002","article-title":"Steroid signalling in the ovarian surface epithelium","volume":"16","author":"Rae","year":"2005","journal-title":"Trends Endocrinol. Metab."},{"key":"ref_52","first-page":"255","article-title":"Ovarian surface epithelium: Biology, endocrinology, and pathology","volume":"22","author":"Auersperg","year":"2001","journal-title":"Endocr. Rev."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"711","DOI":"10.1016\/S0015-0282(16)58201-6","article-title":"Ovarian response to recombinant human follicle-stimulating hormone in luteinizing hormone depleted women: Examination of the two cell, two gonadotropin theory","volume":"65","author":"Gotlieb","year":"1996","journal-title":"Fertil. Steril."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1159\/000292262","article-title":"Are Estradiol-Producing Cells Incompletely Endowed? A Chronicle of the Emergence of Certitude from Conjecture","volume":"41","author":"Lieberman","year":"1996","journal-title":"Gynecol. Obstet. Investig."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1071\/RD01056","article-title":"Lactational control of reproduction","volume":"13","author":"Mcneilly","year":"2002","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.ygcen.2010.07.015","article-title":"Progesterone and reproduction in marsupials: A review","volume":"170","author":"Bradshaw","year":"2011","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1071\/RD9960661","article-title":"Hormones of oestrus and ovulation and their manipulaiton in marsupials","volume":"8","author":"Hinds","year":"1996","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1016\/S0141-1136(02)00169-1","article-title":"Direct comparison of enzyme histochemical and immunohistochemical methods to localize an enzyme","volume":"54","author":"Noorden","year":"2002","journal-title":"Mar. Environ. Res."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1053\/j.semdp.2024.05.001","article-title":"Review of immunohistochemistry techniques: Applications, current status, and future perspectives","volume":"41","author":"Mebratie","year":"2024","journal-title":"Semin. Diagn. Pathol."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"1474","DOI":"10.1210\/en.2016-1851","article-title":"The steroid metabolome in the isolated ovarian follicle and Its response to androgen exposure and antagonism","volume":"158","author":"Lebbe","year":"2017","journal-title":"Endocrinology"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1354\/vp.42-4-405","article-title":"Technical aspects of immunohistochemistry","volume":"42","year":"2005","journal-title":"Vet. Pathol."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1093\/biolre\/ioad098","article-title":"The koala (Phascolarctos cinereus) prostate: A comprehensive histological and immunohistochemical investigation","volume":"109","author":"Campbell","year":"2023","journal-title":"Biol. Reprod."}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/1\/47\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T06:50:06Z","timestamp":1766991006000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/1\/47"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,27]]},"references-count":62,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["biology15010047"],"URL":"https:\/\/doi.org\/10.3390\/biology15010047","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,27]]}}}