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

Abstract

Using the domestic cat as a non-rodent, larger animal model, the objective of the present study 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 the regulation of folliculogenesis. In Study 1: Secondary or early antral follicles encapsulated in 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 their 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 (Steroidogenesis genes)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 ‘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 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.

The mutual benefits of research in wild animal species and human-assisted reproduction

“…However, the follicle culture strategy still needs to be improved using larger-sized animal models than the mouse. Using cat and dog tissues, encouraging results of in vitro culture methods for early-stage follicles were obtained [18,69]. Other cutting-edge approaches also are investigated in carnivores such as the preservation of the GV at room temperature [70].…”

Determination of the appropriate concentration of sodium alginate used for in vitro culture of cat preantral follicles in a serum‐free medium containing FSH, EGF and IGF‐I

“…For this reason, it is essential to determine the adequate concentration of alginate that simultaneously maintains follicle structure during folliculogenesis in the feline species, providing support and permeability to the follicle, and thus ensures an adequate supply of nutrients, hormones and local regulatory factors towards the encapsulated follicle (Xu et al, 2009). In cat, preantral follicles have been cultured either in 2D (Jewgenow & Göritz, 1995; Wongbandue et al, 2013) or alginate‐based 3D culture systems with 0.3% (Nagashima et al, 2021) or 0.5% (Chansaenroj et al, 2019; Songsasen et al, 2012; Songsasen, Thongkittidilok, et al, 2017; Thongkittidilok et al, 2018) of this compound based on previous concentrations used in dogs (Songsasen, Nagashima, et al, 2017).…”

In vitro growth of the ovarian follicle: taking stock of advances in research

“…Follicles produced in vitro are small compared to follicles produced in vivo ( Xiao et al ., 2015 ; Rodrigues et al ., 2015 ). Rodents and mammals are the most prevalent models, approximately one-fifth of the studies use human follicles ( Xiao et al ., 2015 ; Telfer et al ., 2008 ), and other mammalian follicles, like the Rhesus monkey ( Rodrigues et al ., 2015 ; Xu et al ., 2009a ; Peluffo et al ., 2010 ; Xu et al ., 2011a ; Hornick et al ., 2012 ; Xu et al ., 2013 ; Xu et al ., 2015b ; Xu et al ., 2018 ; Baba et al ., 2017 ); baboon ( Xu et al ., 2011b ); bovine ( Yamamoto et al ., 1999 ; Rossetto et al ., 2013a ; b ; Araújo et al ., 2015 ); ovine ( Arunakumari et al ., 2010 ; Muruvi et al ., 2005 ); caprine ( Rossetto et al ., 2013a ; Ferreira et al ., 2018 ; Silva et al ., 2015 ; Magalhães et al ., 2011 ); swine ( Hirao et al ., 1994 ; Wu et al ., 2001 ); cats ( Songsasen et al ., 2017 ; Thongkittidilok et al ., 2018 ); dogs ( Songsasen et al ., 2011 ); horses ( Haag et al ., 2013 ); wildcats ( Wiedemann et al ., 2013 ).…”

“…In mice, for example, collagenase leads to the production of preantral granulosa cell-oocyte complexes (PGOCs) and cell-oocyte complexes (COCs) from ovarian tissues, rather than whole follicles. In the mechanical separation method, special needles are used to separate the follicles of the ovarian stroma or tissue grinders, homogenizers, and cell strainers ( Songsasen et al ., 2017 ; Mahalingam et al ., 2016a ; b ; Craig et al ., 2010 ). The mechanical separation method results in less damage to the follicle than the enzyme method, and provides improved protection to the theca layer and follicular morphology ( Araújo et al ., 2014a ; b ), but the worst problem is that this method takes a lot of time ( Demeestere et al ., 2002 ).…”