An Acad Bras Cienc (2021) 93(2): e20190841 DOI 10.1590/0001-3765202120190841 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal CELLULAR AND MOLECULAR BIOLOGY Gametogenesis and reproductive Running title: GAMETOGENESIS dynamics of Scinax acuminatus (Anura: AND REPRODUCTIVE DYNAMICS OF Scinax acuminatus Hylidae): morphological, histological and immunohistochemical analysis Academy Section: CELLULAR AND MOLECULAR BIOLOGY GABRIELA OLEA, ESTEBAN CHEIJ, ANA PAULA C. BOCCIONI, FLORENCIA RODRIGUEZ, JORGE CÉSPEDEZ & DANIEL LOMBARDO e20190841 Abstract: To characterize the reproductive biology of Scinax acuminatus and contribute to the natural history of this species, the morphology of the reproductive system of males and females was analyzed at anatomical, histological and immunohistochemical 93 levels. The individuals were collected fortnightly between August and December (2) (2016) and January to December (2018). The anatomy of the reproductive system was 93(2) analyzed in a stereoscopic microscope, and histological preparations staining with DOI hematoxylin-eosin and Masson’s trichromic, PAS and Coomassie Blue was performed 10.1590/0001-3765202120190841 as well. To characterize the meiotic-active cells in the testes, immunostaining with the PCNA proliferation protein was performed. There were found females with ovaries with oocytes in different stages of maturity and post-ovulatory females. Males presented continuous spermatogenesis, which could be confirmed by the immunostaining of PCNA in spermatogonia during the cycle. The results of this work serve as a basis for the characterization of the reproductive cycle in S. acuminatus and provide background information on the analysis of spermatogenic activity by IHQ from the study of the immunodetection of the PCNA cell proliferation protein. Future studies will focus on the evaluation of cell death processes during the reproductive cycle in the studied species to compare with those obtained in terms of cell proliferation. Key words: amphibians, ovaries, reproduction, testicles. INTRODUCTION In recent years, the reproductive biology of Neotropical anurans has been studied in Anuran amphibians have a great diversity of Argentine in some species such as: Rhinella reproductive modes among vertebrates (Haddad bergi (Schaefer & Kehr 2006), Leptodactylus & Prado 2005, Wells 2007, Crump 2015); this chaquensis (Schaefer et al. 2006) Scinax complexity is due to the fact that the reproduction nasicus (Hamman et al. 2009), Argenteohyla strategies of the species are determined by the siemersi pederseni (Cajade et al. 2010), Boana combination of morphological, physiological, pulchella and Boana punctata (Antoniazzi et al. behavioral and environmental factors (Toledo 2019); and with emphasis on folliculogenesis & Haddad 2005, Wells 2007, Curi et al. 2014). and spermatogenesis in Ceratophrys ornata Generally, tropical and subtropical species have (Carezzano et al. 2013), Dendropsophus sanborni continuous cycles, while temperate species (Curi et al. 2014), Hypsiboas punctatus (Brunetti have discontinuous or potentially continuous cycles (Haddad & Prado 2005). An Acad Bras Cienc (2021) 93(2) GABRIELA OLEA et al. GAMETOGENESIS AND REPRODUCTIVE DYNAMICS OF Scinax acuminatus et al. 2014), Rhinella arenarum (Scaia 2015, Scaia This work aims to provide data on the et al. 2019). reproductive biology of S. acuminatus, It has been recently proposed that in the from the morphological, histological, and toad cell proliferation in germline is higher immunohistochemical analysis of the during the reproductive season and that cysts in reproductive system in males and females. This late stages of spermatogenesis (spermatocytes would provide more information on gonadal and spermatids) are removed from the testes structures for the later comparison of the by apoptosis immediately after the breeding, reproductive system of other anuran species of suggesting that according to different the same family. environmental or physiological cues (Scaia 2015). The proliferation of germline has been studied in R. arenarum (Scaia 2015, Scaia et MATERIALS AND METHODS al. 2019) and, in more detail, in Pelophylax Study area esculentus (Ferrara et al. 2010). In P. esculentus Samplings were carried out every two weeks seasonal variation of proliferations seems to be between August and December 2016 and from associated with changes in the proliferating cell January to December of 2018, 10 km NE near the nuclear antigen PCNA (Chianese et al. 2015). As a city of Corrientes, (Paraje Perichón: 27 ° 26’36.6 consequence, maximum cell proliferation during “S 58 ° 45’14.0” W). Phytogeographically, the the reproductive season is associated with high sampling site is located in the Humid Chaco levels of PCNA. (Cabrera 1976). The climate is subtropical or Although there are studies on reproductive mesothermal (Carnevali 1994), with an average biology in the Hylidae family, there are few who annual temperature gradient that oscillates analyze cell proliferation during the reproductive from north to south between 21 ºC and 19, 5 ºC, cycle with emphasis on gametogenesis. For these with January being the warmest month (annual reasons, the present analysis of the reproductive average, 27 ºC) and July the coldest one (annual biology of S. acuminatus will contribute data on average, 14 ºC). In addition, rains are abundant, gametogenesis during the reproductive cycle frequent and irregular throughout the year, in female and male; and the spermatogenic presenting their minimum level in winter activity of the testicles from the detection of (Bruniard 1999, Carnevali 1994), with an average proliferating cells from immunostaining with annual rainfall of more than 1,500 mm in the PCNA. northeast and 1,000 mm in the southeast of the S. acuminatus (Cope 1862), commonly province. known as chaqueña snout frog, is a more jumping than walker frog related to natural and Methodology for capture and processing anthropic environments, and inhabits humid material areas with low vegetation, near estuaries and The manual capture of a total of 22 individuals of other bodies of water. It is distributed in 7 S. acuminatus was carried out. The animals were provinces of Argentina: Chaco, Corrientes, Entre taken out to the laboratory in plastic bags. Then, Ríos, Formosa, Salta, Santa Fé, and Misiones the euthanasia of the specimens was carried (Zaracho et al. 2012). It is also present in Bolivia, out with a 2% lidocaine overdose, following Brazil, and Paraguay. the protocol established in the Guide for Animal Euthanasia proposed by the IACUC (The An Acad Bras Cienc (2021) 93(2) e20190841 2 | 11 GABRIELA OLEA et al. GAMETOGENESIS AND REPRODUCTIVE DYNAMICS OF Scinax acuminatus Institutional Animal Care and Use Committee) (50-50%) was performed for 24 hours and under the rules of the ethics committee in force pure paraffin overnight. After this, the block- in resolution CIC400-MED-2017. The specimens paraffins were made, and the samples were collected are in the didactic collection of the oriented to obtain transverse or longitudinal Laboratory of Herpetology - UNNE. sections of 5 to 7 microns. These were obtained with a Spencer manual rotary microtome. The Morphological analysis of the gonadal system samples were stained with Hematoxylin-Eosin The snout-cloaca length of each specimen was (H-E) and Masson’s Trichromic for general recorded with a precision caliper of 0.01mm. cytological and histological characterization. For the macroscopic analysis of the gonadal At the histochemical level, PAS (Periodic acid- system, we proceeded to the dissection of the Schiff-haematoxylin) reactions were applied for specimens and subsequent observation under the detection of neutral glycosaminoglycans a stereoscopic microscope, and isolation of the (Kiernan 1999), and Coomassie blue staining reproductive system. In the case of males, both was performed for the determination of testicles were taken, and the maximum length proteins. In the case of males, samples with and width were measured to estimate the average spermatozoa in the seminiferous tubules were size of the gonads with a digital caliber of 0.01mm considered mature. The images were captured precision. In females, the presence and number using the LEICA DM4000B microscope, and an of follicles were analyzed, differentiating them image capture system supported by the LASZ by type. For the determination, the classification LEICA Inc® program. The camera is coupled to a proposed by Valdez Toledo & Pisanó (1980) was trinocular microscope model LEICA® DCC-380X®. followed: Previtellogenic oocytes: translucent, with no accumulation of vitelline platelets in Immunohistochemical studies to determine the cytoplasm; vitellogenic oocytes: whitish the immunodetection of PCNA coloration without pigmentation, with onset of The immunodetection of PCNA was only analyzed vitellogenesis; post-vitellogenic oocytes: greater in testicles because it wanted to highlight the than the previous ones, with well-differentiated possibility of detecting a continuous cycle animal and vegetal poles. The presence-absence without the need to analyze the presence of post-ovulatory bodies was also recorded. The or absence of sperm. In the case of females, mature females were identified by the presence this analysis was not necessary given that the of post-vitellogenic oocytes. presence of follicles in different phases of folliculogenesis corroborates