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Abstracts. II International Symposium on Animal Biology of Reproduction, Nov. 19-22, 2008, São Paulo, SP, Brazil. Apoptosis changes in the ovaries of rats submitted to feed restriction during embryonic development T.T. Zampieri 1 , F.C. Braga, F.V. Meirelles 2 , C.L.V. Leal 1 1 Laboratory of Histology, Dept. of Basic Sciences, FZEA, Pirassununga, SP, Brazil. 2 Laboratary of Molecular Morphophysiology and Development, Dept. of Basic Sciences, FZEA, Pirassununga, SP, Brazil. Introduction Delayed ovary development during the embryonic period has been shown to result from mal-nutrition during pregnancy. As a result there is a decrease in pre-antral follicles formation that can lead to fertility problems at adulthood (1), such as changes in follicular atresia and the expression of apoptosis genes regulators, which are an important component of embryonic development and follicular function until later in life (2). The Bcl-2 gene family members are involved in cell proliferation, follicular selection and luteolysis, and the ratio between Bcl-2 (prosurvival molecule)/BAX (pro-apoptotic molecule) shows to be critical for the normal survival of germ cells (the higher the ratio the lower the apoptosis). This study aimed to establish the relative expression of apoptosis genes mRNA in the ovaries of rats whose mothers were submitted to feed restriction during pregnancy and verify the efficacy of early dietetic correction after parturition on such expression. Materials and Methods During pregnancy 24 female Wistar rat (Rattus novergicus) were divided into two groups: GI, received free-feeding diet; GII received 70% of normal daily consumption. GI was considered the control group and GII experimentally produced the malnutrition during pregnancy. After parturition, during lactation, the GII females received freefeeding diet similar to GI, to establish the dietetic correction. The offspring ovaries were submitted to histological analysis (embedded in resin and stained with hematoxilin-eosin) and quantification of relative mRNA abundance for Bcl-2 and BAX (RT-PCR) at birth, weaning and puberty. Results and Discussion No difference in the number of oocytes/mm 2 (18 x 10 3 ) between groups at birth (p > 0.05). There was a difference in the proportion of pre-antral follicle (GI = 26.17%; GII = 44.07%, p = 0.053) and large-antral follicles (GI = 26.12%; GII = 16.67%, p=0.028) between treatments at weaning. On the other hand, the proportion of small-antral follicles was the same between GI and II (GI = 47.71%; GII = 39.27%, p = 0.923). At puberty, the animals did not present differences (p > 0.05) in the different follicle categories (pre-antral follicles = 37.17%, small-antral = 29.62%, largeantral = 33.23%). Regarding the relative gene quantification of Bcl-2 and BAX, a difference between experimental groups was observed only at weaning (Fig. 1), with GI showing higher relative expression of apoptotic genes (GI = 2.26 ± 0.82; GII = 0.51 ± 0.30, p = 0.024). Animals from GI presented a lower Bcl-2/BAX ratio at birth (suggesting higher apoptosis), that tended to increase and stabilize from weaning. On the other hand, GII at weaning presented a lower Bcl-2/BAX ratio that increased to normal values at puberty. It is suggested that higher expression of BAX from GII weaning animals could occur due to the inferior number of viable follicles (2). Feed restriction during pregnancy was sufficient to cause an unbalance on follicular population and on expression of genes related to apoptosis. Finally, the early dietetic correction (from birth) reverted the alterations when the animals reached puberty. Figure 1. Ratio of relative expression of Bcl-2/BAX of control (GI) and feed restricted (GII) groups at birth, weaning and puberty. References (1) Rhind SM, 2004. Anim Reprod Sci, 82/83:169-181; (2) Lea RG et al. 2006. Reproduction, 131:113-124. Financial support: FAPESP E-mail: thaizampieri@gmail.com Anim. Reprod., v.6, n.1, p.239, Jan./Mar. 2009 239
240 Abstracts. II International Symposium on Animal Biology of Reproduction, Nov. 19-22, 2008, São Paulo, SP, Brazil. Immunolocalization of proteins involved in the signal transduction mechanism of p-ERK ½ in canine prostates with different diagnoses of Benign Prostate Hyperplasia K.S. Oliveira 1 , J.F. Pérez-Gutiérrez 2 , R.L. Amorim 3 , J. Oliva-Hernández 4 , G.H. Toniollo 1 1 Dept. of Preventive Veterinary Medicine and Animal Reproduction, FCAV/UNESP, Jaboticabal, São Paulo, 14884-900Brazil, 2 Dept. of Animal Medicine and Surgery, FV/UCM, Madrid, 28040, Spain, 3 Dept. of Veterinary Clinics/FMVZ/UNESP, Botucatu, SP, 186180-000, Brazil, 4 INIFAP, Tabasco, 86400, México. Introduction The canine prostate gland is the only accessory gland of the genital apparatus of this species and it is a locus of several pathological processes in middle-aged to senile animals (1), such as Benign Prostate Hyperplasia (BPH). The proliferation, differentiation, and organization of animal tissue cells are controlled by various forms of intercellular communication. One of these forms involves a network of polypeptides such as growth factors, which activate membrane receptors for the transduction of proliferation and differentiation signals (2). The aim of this study consisted in immunolocating the proteins CYR61, EGF, TGF-α, EGFr, and p-ERK1/2 via immunohistochemistry (IHC) in canine prostates with by different BPH diagnoses. Material and Methods Twenty-three prostate fragments were investigated, of which 5 were considered normal, 6 were diagnosed with benign prostate hyperplasia of the epithelial type (ep-BPH), 6 with benign prostate hyperplasia of the stromal type (s-BPH), and 6 with complex benign prostate hyperplasia (CBPH). All fragments were assessed by the IHC technique (3) for the polyclonal antibodies (and respective concentrations) anti-CYR61 (1/400), anti-EGF (1/20), and anti-p-ERK1/2 (1/200) as well as for the monoclonal antibodies anti-TGF-α (1/20) and anti-EGFr (1/20). Secondary polyclonal and monoclonal biotinilate antibodies (Vectastain Elite Kit ABC – Vector laboratories, Burlingame, CA, USA) followed the concentration pattern indicated by the manufacturer. Lamina treated with polyclonal and monoclonal antibodies were assessed under optical microscopy with regards to the presence and absence of marking in stromal components (str. c.) - smooth muscle fibers (SMF), fibroblasts (F), smooth musculature of arteries and/or arterioles (SMA), endothelial cells (EC), and mononuclear cell infiltrate (MC) - and epithelial acini (epith. c.), such as cytoplasm and nucleus of secretory cells. Results and Discussion All investigated fragments (n = 23) revealed a positive marker for CYR61, EGF, TGF-α, EGFr, and p-ERK1/2 proteins. Stromal and epithelial acini components also revealed a positive marker in various levels of intensity. Only the normal fragments failed to show a significant marker in stromal components for TGF-α and EGFr proteins (Table 1). Table 1. Stromal and epithelial components which revealed the most prominent protein markers in each diagnosis of canine prostate under study. Diagnoses Protein Normal Ep-BPH S-BPH CBPH Str.C Epith.C. Str.C. Epith.C. Str.C. Epith.C. Str.C Epith.C. CYR61 SMF, SMA nucleus SMF, SMA nucleus SMF, SMA cytoplasm SMF, F nucleus EGF SMF, F nucleus SMF nucleus FML nucleus SMF, EC nucleus TGF-α X cytoplasm SMF, MC cytoplasm MC, SMF, F cytoplasm EC cytoplasm EGFr X nucleus SMF, SMA nucleus SMF, F nucleus SMF, F nucleus p-ERK 1/2 SMF, F nucleus SMF nucleus SMF, F nucleus SMF, EC nucleus Research on proteins involved in cellular differentiation and proliferation has been applied to several tissue alterations within veterinary medicine. The comparison of the immunolocalization of these proteins in both normal and altered tissues is of vital importance for the knowledge of their actions, for the establishment of signal transduction routes, and for the production of data applied to pharmacological studies in order to block the superexpression and/or activation of such proteins and thus reduce chances of neoformations of altered tissues. References (1) Krawiec DR, Heflin D. 1992. Study of prostatic disease in dogs: 177 cases (1981-1986). J Am Vet Med Assoc, 200:1119-1122; (2) Massagué J. 1987. The TGF-β family of growth and differentiation factors. Cell, 4:437-8; (3) Oliveira KS, Araújo EG Menezes LB, Damasceno AD, Fioravanti MCS, Amorim RL. 2006. CYR61, a cellular proliferation marker in dog with prostatic disease. Theriogenology, 66:1618-1620. Financing agencies: FAPESP, CAPES. E-mail: ksoliver13@hotmail.com. Anim. Reprod., v.6, n.1, p.240, Jan./Mar. 2009
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