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Abstracts. II International Symposium on Animal Biology of Reproduction, Nov. 19-22, 2008, São Paulo, SP, Brazil.
Identification of IGF-II from mRNA in Boer bucks spermatozoa
I.J. Arruda 1
, G.M.L. Holanda 3
, F.Q.G. Bezerra 2 , C.R. Aguiar-Filho 2 , I.M.T. Lima 1
, A.L. Souza 1
,
M. Adrião 3 , M.A.L. Oliveira 2 , D. Rondina 1
1 Laboratório de Nutrição e Produção de Ruminantes, UECE/FAVET, Fortaleza, CE, Basil; 2 Laboratório de Reprodução da
Faculdade de Medicina Veterinária, UFRPE; 3 Laboratório Fisiologia Animal Molecular Aplicada, FAMA, UFRPE, Recife, PE,
Brasil.
Introduction
Evaluation of protein pattern in spermatozoa is a crucial step to identify their role in the molecular regulation of
reproductive mechanisms. In humans (1), cattle (2) and other species (3), IGF-II has been identified in spermatozoa
and was positively correlated with semen quality. Thus, the aim of this study was to identify the IGF-II in the Boer
bucks spermatozoa.
Materials and Methods
Fifty semen samples were collected from six adult Boer bucks with normal seminal parameters according to (4).
Spermatozoa were separated from seminal plasma by centrifugation (15000 rpm/15’) and stored in -20 °C until
RNA extraction. In twenty-five samples, spermatozoa were capacitated with swim-up method according to (5). The
total RNA extraction was initiated transferring the pellet to a new tube, in which was added Trizol, and,
subsequently, the mixture was incubated for 5 minutes at room temperature. After digestion, aqueous phase was
extracted with chloroform and the RNA precipitation was performed using isopropilic alcohol in a proportion of 1:1.
The pellet was washed with ethanol 75%, dried at ambient temperature for 15 minutes, resuspended in DEPC water
and stored at – 80 °C. The total RNA samples were treated with DNAse. To detect IGF-II in spermatozoa mRNA,
RT-PCR and electrophoresis in gel-agarosis was performed in agreement with (6).
Results and Discussion
The primers used were based on the bovine sequence of IGF-II gene (P07456). Amplifications procedure showed a
product of 63,5 bp (Fig 1). The presence of IGF-II was verified respectively, in each buck, in the capacitated
spermatozoa and in the spermatic pool (Fig 1). Also, from all spermatic RNA samples, it was isolated a PCR
product with similar molecular weight. These results are in agreement with the studies carried out in humans (6, 7).
In conclusion, our findings showed that IGF-II is present both in non-capacitated sperm (individually or in pool), as
the capacitated buck sperm.
Figure 1. RT-PCR of IGF-II in the goat spermatozoa. Well 1(MA): molecular marker of 50bp, Well 2 (C+): positive
control – GAPDH, Well 3(S.I): semen sample for individual , Well 4(S.C): capacitated spermatozoa, Well 5(P.O):
sperm pool, Well 6(C-): negative control.
References
(1) Glander HJ et al. 1996. Hum Reprod, 11:2454-2460; (2) Henricks DM et al. 1998. Biol Reprod, 59:330-337; (3)
Naz RK, Padman P. 1999. Arch Androl, 43:153-159; (4) Manual para exame andrológico e avaliação de sêmen
animal. 1998. 2. ed. Belo Horizonte: CBRA. (5) Sánchez-Luengo et al. 2005. Fertil Steril, 83:1064-1066; (6) Aquila
S et al. 2005. Endocrinology, 146:552-557; (7) Goodwin LO et al. 2000. Mol Hum Reprod, 6:127-136.
E-mail: iracelmavet@yahoo.com.br
50bp
51,5pb GAP DH
Anim. Reprod., v.6, n.1, p.241, Jan./Mar. 2009 241