Short-term hypertonic exposure enhances in vitro follicle growth and meiotic competence of enclosed oocytes while modestly affecting mRNA expression of aquaporin and steroidogenic genes in the domestic cat model.
Using the domestic cat as a non-rodent, larger animal model, the objective was to determine the impact of a brief incubation in a hypertonic microenvironment on (1) ovarian follicle and oocyte growth in vitro, (2) developmental capacity of the resident oocyte, and (3) expression of aquaporin (AQP) genes in parallel with genes involved in regulation of folliculogenesis. In Study 1: Secondary or early antral follicles encapsulated in 0.5% alginate were allocated to one of three treatment groups: 1) culture in standard medium at 290 mOsm for 15 d (Control); 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for 15 d (Hypertonic-1h); or 3) incubation in 350 mOsm medium for 24 h followed by incubation in standard medium for additional 14 d (Hypertonic-24h). After measuring follicle and oocyte diameters on Day 15, in vitro-grown oocytes were incubated for 24 h before assessing nuclear status. In Study 2: secondary or early antral follicles were subjected to one of the three treatments: 1) culture in standard medium at 290 mOsm for 48 h; 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for additional 47 h; or 3) incubation in 350 mOsm medium for 24 h followed by culture in standard medium for additional 24 h. At the end of the culture period, all follicles were assessed for mRNA level of Cyp17a1, Cyp19a1, Star, Aqp1, 3, 5, 7 and 8 as well as Fshr using qPCR. Freshly collected follicles also were subjected to gene expression analysis and served as the 'Non-cultured control'. Hypertonic-24h follicles grew larger (P < 0.05) than the control, whereas those in Hypertonic-1h group exhibited intermediate growth, especially when the culture started at the early antral stage. Oocytes in the Hypertonic-24h group were larger and resumed meiosis at a higher rate than in the other treatments. In vitro culture affected (P < 0.05) mRNA expression of Cyp19a1, Star, Aqp1, and Aqp7 in both the secondary and early antral stage while Fshr was only affected in the former compared to the non-cultured control. Pre-incubating follicles in 350 mOsm medium for 24 h enhanced (P < 0.05) Star and Aqp7 while decreasing (P < 0.05) Aqp1 expression compared to the control in secondary follicles, but not in the early antral stage. In summary, short-term hypertonic exposure promoted cat follicle development in vitro (including the meiotic competence of the enclosed oocyte) possibly through a mechanism that does not involve water transport genes.
Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, 22630, USA. Electronic address: songsasenn@si.edu.
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Songsasen
Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, 22630, USA.
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Thongkittidilok
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
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Yamamizu
Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, 22630, USA.
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Wildt
Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, 22630, USA.
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Comizzoli
D000818:Animals; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D001141:Aromatase / Q000235:genetics / Q000378:metabolism*; D002415:Cats; D018929:Cell Culture Techniques / Q000662:veterinary; D000072478:Cytochrome P450 Family 17 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D006982:Hypertonic Solutions / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D006080:Ovarian Follicle / Q000187:drug effects* / Q000254:growth & development*; D012333:RNA, Messenger / Q000378:metabolism; D011962:Receptors, FSH / Q000235:genetics / Q000378:metabolism*
D020346:Aquaporins; D006982:Hypertonic Solutions; D012333:RNA, Messenger; D011962:Receptors, FSH; D001141:Aromatase; D000072478:Cytochrome P450 Family 17
Cat; Gene expression; Hypertonic exposure; In vitro follicle culture; Meiotic maturation