Expression of Markers Associated with Ovarian Cycle in Bitches

Introduction - Although the hormonal changes during estrus cycle of bitches are well- known, knowledge in luteal phase and anestrus are incomplete. Information on phenomenon perform in cytosol during estrous cycle, especially in bitch are extremely rare. The crosstalk among the different signals determines the fate of the ovarian follicle. Because there are multiple paracrine and autocrine signals, it is still not completely understood which are the critical factors that discriminate between the follicles destined for elimination by apoptosis (the major population and the follicles) and between few of the follicles that will continue to develop to reach the final stage of a Graafian preovulatory follicle. Progesterone formation and release during the estrous cycle may play an important role in the fertilization of the oocyte during ovulation. It has been suggested that early progesterone production in the preovulatory follicle impairs the quality of the mature egg during fertilization (Lindheim et al 1998, Fanchin et al 1997, Urman et al 1999). In contrast, proper timing of progesterone production and the duration of its secretion seems to be critical for maintaining functional granulosa-lutein cells, subsequent to the LH surge and maintenance of the corpus luteum during pregnancy. Progesterone interaction with its cytosolic receptor may play a part in the survival activity of the granulosa-lutein cells (Eva et al 2001).

Objectives - Aim of our study is to reveal the connections on receptor and cytosol level behind the hormonal changes during different stage of canine estrous cycle.

Materials and methods - The whole procedure fulfils all the requirements of the European Animal Welfare regulations. Bitches are ranged among groups on basis of stage of cycle. Ovarian tissue is collected from 30 healthy bitches with ovariectomy or ovariohysterectomy for surgical spaying or medical purposes like unwanted pregnancy. Bitches are in different stage of ovarian cycle. Serum samples for hormone determinations are collected before initiation of anaesthesia from cephalic vein. The ovarian tissues are fixed in 5–8% neutral- buffered formalin, dehydrated and embedded in paraffin wax. From each paraffin block 4 μm thick sections are cut and stained with haematoxylin and eosin. The sections are immunostained using the En Vision System with primary mouse antibody. The progesterone antibody is prepared by the laboratory of the Department of Obstetrics and Reproduction, Szent István University, Faculty of Veterinary Science, Budapest, Hungary, and diluted 1:300. Progesterone antibody is produced for use in a quantitative ELISA for the determination of progesterone in canine serum (Siklódi et al., 1995, Thuróczy et al 2007). The Ki-67 (Clone: MIB-1), vimentin (Clone: V9) and pan Cytokeratin antibody (Clone: AE1/AE3) are prepared by DakoCytomation (Glostrup, Denmark). The p53 (Clone: MDM2- SPM344), estrogen receptor  antigens (Rabbit polyclonal to Estrogen) are prepared by Abcam (Cambridge, UK). Serum progesterone, LH, FSH, and 17 -estradiol -levels are determined from the serum. LH concentration is measured by LH Detect® (INRA, Nouzilly, France), FSH concentrations are measured by use of Canine FSH ELISA (AE C004) (Biocode – Hycel, Liege, Belgium), estrogen concentrations are measured by ELISA assay (E2-EASIA, Biosource Europe, Nivelles, Belgium). Progesterone concentration is measured by Quanticheck, validated to dog serum (SzIU, Faculty of Veterinary Science, Budapest) (Nagy et al. 1998.).

Results - Hormone concentrations in proestrus and in oestrus correspond to the preliminary data (PE: P4:5,41 ng/ml; E2:27,86 pg/ml; FSH:7,19ng/ml; OV: P4:6,04 ng/ml; E2:22,81 pg/ml; FSH:8,31 ng/ml) (Johnston et al. 2001, Kooistra et al. 1999). Measured hormone concentrations in the four different phases of metoestrus (ME1-4) differ from our expectations (ME1: P4:2,87; 40,0 ng/ml; E2:56,18; 110,83 pg/ml; FSH:1,45; 3,26 ng/ml; ME2: P4:4,08; 43,0 ng/ml; E2:8,39; 201,53 pg/ml; FSH:13,76; 9,95 ng/ml) (Johnston et al. 2001, Kooistra et al. 1999). The concentration of estrogen and FSH was the same as in the oestrus (E2:18,47 pg/ml, FSH:6,95 ng/ml).
The corpus luteum is present in every stages of estrous cycle, especially in anestrus. Although progesterone is findable in cytoplasm of ovarian cells in each stage, the presence is restricted to follicular cells during the anestrus. Strong reaction is given by AE1/AE3, vimentin and p53 in each estrous stage, in contrast with Ki67, respectively.