Follicular Population and Oestrogen Receptor Alpha in Ovary of the Bitch

DoleÏe l R. , R . Kyl iánková , V. Kummer , J . Ma‰ková , P . S ta rá , R . Vi tásek: Follicular Population and Oestrogen Receptor Alpha in Ovary of the Bitch. Acta Vet. Brno 2004, 73: 37-43. The objective of this study was to evaluate the follicular population on ovaries as related to the phases of oestrous cycle and age in bitches. Several characteristics of follicular population on ovaries in follicular (Group 1, n = 3), luteal (Group 2, n = 7) and anoestral (Group 3, n = 8) phases of the oestrous cycle and in young (≤ 6 years, n = 12) and old (≥ 7 years, n = 6) bitches were observed. The numbers and proportions of primary, secondary, tertiary and atretic follicles, diameters of the follicles and oocytes and distribution of oestrogen receptor alpha in ovaries after ovariectomy were evaluated. Microscopical examination of slices stained with haematoxylin-eosin was performed in 50 mm2 of ovarian tissue in each animal and immunohistochemical detection of oestrogen receptor alpha was performed using a monoclonal oestrogen receptor alpha antibody. Average numbers of all follicles were found 567 (31% atretic), 449 (27% atretic) and 408 (37% atretic) in the Group 1, 2 and 3, respectively. Proportions of primary, secondary and tertiary follicles regardless of phase of the oestrous cycle as well as age of animals were 72.0%, 24.5% and 3.5%, respectively. Proportions of these follicles in various phases of the oestrous cycle as well as in young and old animals were not different. Diameters of primary (40.2 ± 15.20 μm), secondary (102.4 ± 36.60 μm) and tertiary (323.7 ± 95.84 μm) follicles as well as diameters of oocytes in secondary (80.7 ± 16.66 μm) and tertiary (105.8 ± 17.64 μm) follicles increased synchronously to follicular development (p < 0.01). High intensity of nuclear oestrogen receptor alpha immunostaining was found within surface epithelium, in primary follicles and in luteal cells. Medium immunoreaction was seen in secondary follicles and low expression of oestrogen receptor alpha was in tertiary follicles. No immunoreaction of oestrogen receptor alpha was found in atretic follicles. The results show similar characteristics of follicular population in various phases of the oestrous cycle both in young and old bitches. Diameters of follicles as well as diameters of oocytes increased and contrary to this oestrogen receptor alpha expression decreased synchronously to follicle development. The study did not show an effect of oestrous cycle and age on proportions of primary, secondary, tertiary and atretic follicles as well as oestrogen receptor alpha distribution on ovaries of the bitch. Follicle, atretic follicle, oestrogen receptor alpha, oestrous cycle, age, bitch The morphological and functional properties of ovarian follicles represent objective of the recent research in many species of animals because quantity and quality parameters of the follicular population define potency in reproduction. In addition, deeper knowledge of follicular development gives an opportunity to increase efficiency of biotechnological methods in reproduction. However, there is a lack of data about follicular population in bitches. Ovaries in newborn mammals contain several tens or hundreds of thousands of primordial follicles, which represent a pool from which they will be recruited for growth during postnatal life (Baker 1972). About half a million of primordial and primary follicles are present on ovaries of a newborn puppy. Ovarian follicles successively develop and are ACTA VET. BRNO 2004, 73: 37–43 Address for correspondence: Doc. MVDr. Radovan DoleÏel, CSc. Clinic of Ruminant Diseases University of Veterinary and Pharmaceutical Sciences Palackého 1-3, 612 42 Brno, Czech Republic Phone:+ 420 541 562 316 Fax: + 420 541 562 332 E-mail: dolezelr@vfu.cz destroyed during postnatal life and the total number of follicles declines to 35 000 and 500 in the middle and by the end of reproduction activity in bitches. The number of follicles is higher in crossbreeds and there is a positive correlation of total number of follicles with size of the ovary as well as size of the bitch (Durrant et al. 1998). Distribution of ovarian steroid receptors represents qualitative parameter of the ovarian structures because steroid hormones regulate ovarian functions via endocrine, paracrine and autocrine mechanisms (P ineda 2003). In spite of the fact that oestrogen and progesterone receptors have been detected in ovaries of several animal species, only progesterone receptors have been described in canine ovaries (Vermei rsch et al. 2001). Object of this study was the occurrence of ovarian follicles in various developmental stages in young and old bitches as well as in various phases of oestrous cycle, diameters of the follicles and oocytes and distribution of oestrogen receptor alpha in canine ovaries. Materials and Methods Experimental animals and samples Eighteen bitches of various breeds between 9 months and 11 years of age, presented for ovariectomy, were used in the study. Stages of oestrous cycle were determined on the basis of anamnesis, clinical examination, vaginal cytology and blood progesterone analysis performed before ovariectomy. Three experimental groups were established according to the phase of oestrous cycle – Group 1 (follicular phase – pro-oestrus and oestrus, n = 3), Group 2 (luteal phase dioestrus, n = 7) and Group 3 (anoestral phase anoestrus, n = 8). In addition the bitches were divided by age – young (≤ 6 years, n = 12) and old (≥ 7 years, n = 6). Thirty six ovaries were visually evaluated immediately after ovariectomy and they were fixed in 10% neutral buffered formalin until laboratory examination. Laboratory examinat ion Six slices from each ovary were paraffin-embedded and then sections were made and stained with haematoxylin and eosin for histological examination. Immunohistochemical detection of oestrogen receptor alpha (ERα), carried out in paraffin sections, was preceded by heat-induced antigen retrieval in a pressure cooker. The prediluted monoclonal anti-ERα antibody 1D5 and the visualization system EnVisionTM+ (Dako, USA) with 3,3 ́ diaminobenzidine (Fluka Chemie, Germany) as the chromogen were used in accordance with instructions of the manufacturers. Small oocyte enclosed by a single layer of cuboidal follicular cells, oocyte with several layers of follicular cells (as case may be vesicles among follicular cells), and oocyte in distinct uniform cavity surrounded by multiple layers of differentiated follicular cells were classified as primary, secondary and tertiary follicles, respectively. Irregularity and reduction of layers of the follicular cells and symptoms of cell degeneration were considered as symptoms of follicle atresia. Numbers of non-atretic as well as atretic primary, secondary and tertiary follicles were determined in 20 visual areas (total area = 50 mm2) in each animal. Follicle diameters were measured in 200 follicles of each developmental stage and oocyte diameters were measured in 100 oocytes in secondary as well as tertiary follicles. Follicle and oocyte diameters and intensity of ERα immunostaining were determined by computer image analysis using the Lucia G system (Laboratory Imaging, Czech Republic). Stat is t ical evaluat ion T-test for Paired Comparison was used for evaluation of follicular population in various phases of the oestrous cycle as well as for diameters of follicles and oocytes. Follicular populations in young and old bitches were compared by means of Chi-Square Test or Fisher ́s Exact Test.

destroyed during postnatal life and the total number of follicles declines to 35 000 and 500 in the middle and by the end of reproduction activity in bitches.The number of follicles is higher in crossbreeds and there is a positive correlation of total number of follicles with size of the ovary as well as size of the bitch (Durrant et al. 1998).
Distribution of ovarian steroid receptors represents qualitative parameter of the ovarian structures because steroid hormones regulate ovarian functions via endocrine, paracrine and autocrine mechanisms (Pineda 2003).In spite of the fact that oestrogen and progesterone receptors have been detected in ovaries of several animal species, only progesterone receptors have been described in canine ovaries (Vermeirsch et al. 2001).
Object of this study was the occurrence of ovarian follicles in various developmental stages in young and old bitches as well as in various phases of oestrous cycle, diameters of the follicles and oocytes and distribution of oestrogen receptor alpha in canine ovaries.

Experimental animals and samples
Eighteen bitches of various breeds between 9 months and 11 years of age, presented for ovariectomy, were used in the study.Stages of oestrous cycle were determined on the basis of anamnesis, clinical examination, vaginal cytology and blood progesterone analysis performed before ovariectomy.Three experimental groups were established according to the phase of oestrous cycle -Group 1 (follicular phase -pro-oestrus and oestrus, n = 3), Group 2 (luteal phase -dioestrus, n = 7) and Group 3 (anoestral phase -anoestrus, n = 8).In addition the bitches were divided by age -young (≤ 6 years, n = 12) and old (≥ 7 years, n = 6).Thirty six ovaries were visually evaluated immediately after ovariectomy and they were fixed in 10% neutral buffered formalin until laboratory examination.

Laboratory examination
Six slices from each ovary were paraffin-embedded and then sections were made and stained with haematoxylin and eosin for histological examination.Immunohistochemical detection of oestrogen receptor alpha (ERα), carried out in paraffin sections, was preceded by heat-induced antigen retrieval in a pressure cooker.The prediluted monoclonal anti-ERα antibody 1D5 and the visualization system EnVision TM + (Dako, USA) with 3,3´diaminobenzidine (Fluka Chemie, Germany) as the chromogen were used in accordance with instructions of the manufacturers.
Small oocyte enclosed by a single layer of cuboidal follicular cells, oocyte with several layers of follicular cells (as case may be vesicles among follicular cells), and oocyte in distinct uniform cavity surrounded by multiple layers of differentiated follicular cells were classified as primary, secondary and tertiary follicles, respectively.Irregularity and reduction of layers of the follicular cells and symptoms of cell degeneration were considered as symptoms of follicle atresia.Numbers of non-atretic as well as atretic primary, secondary and tertiary follicles were determined in 20 visual areas (total area = 50 mm 2 ) in each animal.Follicle diameters were measured in 200 follicles of each developmental stage and oocyte diameters were measured in 100 oocytes in secondary as well as tertiary follicles.Follicle and oocyte diameters and intensity of ERα immunostaining were determined by computer image analysis using the Lucia G system (Laboratory Imaging, Czech Republic).

Statistical evaluation
T-test for Paired Comparison was used for evaluation of follicular population in various phases of the oestrous cycle as well as for diameters of follicles and oocytes.Follicular populations in young and old bitches were compared by means of Chi-Square Test or Fisher´s Exact Test.

Results
Total number of ovarian follicles found in 50 mm 2 of ovarian tissue was 567 (31% atretic), 449 (27% atretic) and 408 (37% atretic) in Groups 1, 2 and 3, respectively.A higher total number of follicles and a lower rate of atresia were found in young bitches compared to the older ones (1321 : 999, p < 0.01 and 36% : 51%, p < 0.001).Proportions of primary, secondary, tertiary follicles regardless of the phase of oestrous cycle as well as age of animals were 72.0%, 24.5% and 3.5%, respectively.Proportions of these follicles in various phases of the oestrous cycle as well as in young and old animals were similar (Fig. 1, 2).
Diameters of follicles increased synchronously to follicle development (Table 1) and diameters of oocytes in tertiary follicles were higher in comparison with oocytes in secondary follicles (Table 2).
High and moderate intensities of nuclear ERα immunostaining were determined in surface epithelium as well as in follicular cells of primary and smaller secondary follicles in all groups (Plate V, Fig. 3).The same intensity was found in luteal cells of corpora lutea in Group 2 (Plate V,  Diameters

Discussion
Primordial follicles in canine ovaries with a diameter of 25 µm which were characterized by single layer of follicle cells (Durrant et al. 1998) are comparable to primary follicles with diameter 40.2 ± 15.2 µm in our study.Preantral and advanced preantral follicles (diameter 78 ± 15 and 211.4 ± 1.5 µm) in that study would be compared with small and large secondary follicles but we evaluated only one group of secondary follicles with diameter 102.4 ± 36.6 µm.Early antral follicles with diameter 360.5 ± 18.8 µm corresponded to tertiary follicles in our study (diameter 323.7 ± 95.8 µm).Durrant et al. (1998) describe advanced preantral follicles as a unique group of canine follicles characterized by fully grown, lipid-filled oocyte within a preantral-like granulosa complex.The acquisition of dense cytoplasmic lipid during growth of canine oocytes has been described by Tesoriero (1982).
Advanced preantral follicles with diameters of about 210 µm which contain oocytes 115 µm in diameter (Bolamba et al. 2002) as antral follicles (Hewitt et al. 1998;Hewitt and England 1999;Otoi et al. 2000b) were used for in vitro maturation (IVM) of bitch oocytes.IVM of oocytes represents an important and complicated stage of in vitro production of embryos especially in bitches, because in contrast to the majority of mammalian species canine oocytes reach metaphase II as far as oviduct within 2-5 days after ovulation (Andersen and Simpson 1973;Concannon et al. 1989;Tsuitsui 1989).Hewitt and England (1997) and Otoi et al. (2000a) demonstrated that more oocytes progressed to metaphase II stage if their sizes were > 100 µm in diameter and donor bitches were 1-6-yearold.Thus diameters of follicles as well as oocytes were useful in selection of suitable follicles for isolation and IVM of oocytes.With respect to the method of follicle evaluation in our study, larger secondary follicles and tertiary follicles are suitable for IVM of oocytes in bitches.
Experimental bitches were devided in two groups by their ages: ≤ 6 years (young bitches) and ≥ 7 years (old bitches) because generally reproductive efficiency decreases after the 7th year of age in bitches.In accordance with this data, the total number of follicles in 50 mm 2 of ovarian tissue was lower and the percentage of atretic follicles was higher in old bitches compared to young ones in our study (999 : 1321 and 51% : 36%).Likewise Durrant et al. (1998) isolated more follicles per gram of ovarian tissue in youngest bitches -donors compared to older ones.In addition the ovaries of bitches 6 month of age or younger yielded a lower percentage of follicles in advanced stage of development but more atretic follicles than other age groups in that study.High rate of follicle atresia in prepubertal bitches can illustrate an acceleration of atresia during the first few moths after birth in a bitch (Andersen and Simpson 1973).We could not evaluate follicular population a short time after birth because prepubertal bitches were not included in the study.
Two types of follicle atresia in bitches were described by Spanel-Borowski (1981).Degenerative changes of oocytes are typical for the first type and a degeneration of granulosa layer is typical for the second type of atresia.The first type predominates in preantral follicles, whereas the second type is typical for antral follicles.Above all, the changes in granulosa complex were used for evaluation of follicle atresia in our study.In spite of similar proportions of primary, secondary and tertiary follicles in various phases of oestrous cycle in our study Durrant et al. (1998) found significant differencies in proportions of isolated follicles in relation to ovary status.They isolated more primary and early secondary follicles in pro-oestrus and metoestrus compared to oestrus, as well as more tertiary follicles in oestrus compared to pro-oestrus.But ovary status did not affect the total number of follicles per gram of ovary in postpubertal bitches.
In spite of that, progesterone and oestrogen receptors have been detected in ovaries of several animal species (K i m and Greenwald 1987ab; Vesanen et al. 1991;Vesanen 1993;Odore et al. 1999) as well as in human ovaries (Iwai et al. 1990) data about oestrogen receptor in canine ovaries are missing.Vermeirsch et al. (2001) described progesterone receptor in various structures of canine ovary.They described an increase in staining intensity for progesterone receptor in follicles synchronously with follicle development.Contrary to this, the staining intensity for oestrogen alpha receptor decreased synchronously with follicle development in this study.No differences in receptor distribution and staining intensity in relation to oestrous cycle, except for corpus luteum and theca externa of tertiary follicles, were found in that study.In accordance with these results, distribution and expression of oestrogen receptor alpha were similar during the various phases of oestrous cycle in our study.Thus concentration of steroid hormones in peripheral blood probably does not infuence oestrogen receptor alpha on canine ovaries.Likewise, Vermeirsch et al. (2001) found little correlation between expression of progesterone receptor in ovary and oestradiol, progesterone or testosterone concentrations in peripheral blood.Probably there is different regulating mechanism for steroid hormone receptors in ovarian structures compared with other sexual organs.It seems that the stage of development is more important for expression of hormone receptors in the follicles than blood concentrations of the corresponding hormones.
The results of our study show similar proportions of primary, secondary, tertiary and atretic follicles in young as well as old postpubertal bitches and similar distribution of oestrogen receptor alpha in canine ovaries in follicular, luteal and anoestral phases of the oestrous cycle.Diameters of follicles as well as diameters of oocytes increase and, on the contrary, oestrogen receptor alpha expression decreases synchronously to follicle development.
Fig. 3. High intensity of nuclear ERα immunostaining in surface epithelium (SE) as well as in primary follicles (PF) and moderate immunoreactivity in secondary follicles (SF).