IMMUNOHISTOCHEMICAL STUDY OF ESTROGEN AND PROGESTERONE RECEPTORS IN DIFFERENT SIZE CLASSES OF OVARIAN FOLLICLES IN DROMEDARY CAMELS

IMMUNOHISTOCHEMICAL STUDY localization OF ESTROGEN AND PROGESTERONE RECEPTORS IN DIFFERENT SIZE CLASSES OF OVARIAN FOLLICLES IN DROMEDARY CAMELS

A.M. Saleh ^*; E.A. Abdelhafez^* and D.R.I. Derar ^**

^* Dept. of Anatomy and Histology, Fac. Vet. Med., Assiut University

^** Dept. Theriogenology, Fac. Vet. Med., Assiut University

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                                            ABSTRACT

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Key words: Immunohistochemistry, ER, PR, Camel ovary.

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The present immunohistochemical study indicated that the expression of ER and PR and the secretion of their specific hormones in the ovary of Arabian camel were well correlated with the reproductive cycle.

INTRODUCTION

Steroid hormones are important regulators of reproductive processes in female mammals. Estrogens and progesterone receptors mediate respectively the action of estrogens and progesterone by regulating transcription target genes. Estrogens possess an intrafollicular action by stimulating in synergic manner with FSH the aromatase activity (Adashi et al., 1982 and Fitzpatrick et aland Richards,. 1992). They increase granulosa proliferation (Palter et al., 2001) and they are essential to GnRH receptor expression in the growing ovarian follicles (Kogo et al., 1999).

Receptors for estrogen (ER) are expressed as 2 structurally related subtypes in mammals, ERα and ERβ, which are encoded by 2 distinct genes. The existence of these 2 subtypes may partly explain the selective action of estrogen in different target tissues and in the same tissue in different physiological statuses (Conneely, et al. 20051). Studies on several species have confirmed the differential distribution of these 2 receptors in the ovary: ERβ was detected mainly in granulosa cells, whereas ERα was detectedlocalized in theca cells, interstitial glands,  stromal cells, and germinal epithelium . [(Pelletie et aland Al-afy. 2000; Van den Broeck et al., 2002; Berisha et al., 2002; Amrozi et al., 2004; Sanchez-Criado et al., 2005 and  Salvetti et al., 2007)] or totally absent [Saunders et al., 1997, Hiroi et al. 1999]. Progesterone plays a major role in controlling ovulationory and pregnancy ([Graham et al., 1997)]. The receptors involved in ovarian function are (PRA) and PRB. The importance of estrogen and progesterone receptors in the female reproductive function was revealed by many authors in different mammalian ovariesis revealed by the infertility of PR knockout mice [Lydon, et al., 1995]. Despite their normal ovarian structure, these mice are unable to ovulate even not after exogene stimulation. There is a shortage in the studies dealing with the role of these receptors and their distribution in the ovarian follicles of she-camel. Therefore, the present study aimed to investigate the normal distribution of estrogen and progesterone receptors in the different classes of the ovarian follicles in the  dromedary sshe-camel inand their relation with the serum and follicular estrogen and progesterone hormonal levelto the phase of the phase of the estrous cycle and corpus luteum of induced ovulation..

Also, the level of these hormones was characterized in the follicular fluid and serum of she-camels during different phases of the follicular cycle as well as during luteal activity.

MATERIALS and METHODS

Collection of samples

Ovaries of 35 adult female one-humped camels (Camelus dromedarius) slaughtered at a Cairo abattoir, were collected immediately after slaughter. The period from November to April was taken as the peak breeding season, while May-October was considered as the low breeding season.

Pre-slaughter information regarding the nutritional or reproductive status of these camels was not available. After cleaning each ovary off the extraneous tissue, diameter of Graafian follicles was measured using Vernier Calipers. On the basis of their size, follicles were classified into three groups viz. small (5-9 mm) and large (10-20 mm). Fluid from each follicle was aspirated aseptically and stored at –20°C. Animals having ovaries with any pathological lesions, or those with cystic follicles (>20 mm in diameter; Tibary and Anouassi, 1996) were not included in the study. Before slaughter, about 15 ml peripheral blood was collected from each animal, serum was separated and stored at –20°C for hormonal analysis.

Histological and immunohistochemsitry:

Fifteen clinically healthy non lactating non pregnant she camels were assigned for the present study. The animals were examined shortly before their scarification oOvaries of tennon-pregnant she camel were collected and at a local abattoir in Cairo governorate, (Egypt ), within 20 minutes of death the ovaries were taking.  The collected ovaries  were conditioned, fixed in 410% neutral buffered formalin for 18-20 hours at 4˚uC, and then washed in phosphate-buffered saline and; processed for in the routine manner; and embeddedin paraffin embedding. Serial sections 5 mm in thickness were mounted on 3-aminopropyl triethoxysilane (Sigma, USA)-coated slides and dried for 24 hours at 37uC. Histological slides of Six 5 µm in thickness slices from each ovary were paraffin-embedded and then sections were made awerend stained with haematoxylin and eosin for histological examination.

For Immunohistochemistry:  An indirect immunohistochemistry method (as described by Salvetti, (2004)) was used. to detect  Antibody against estrogen receptor (ER α) protein and progesterone receptor (PR) protein protein using antibodies obtained from were purchased from( Dako (- Life Trade, Cairo, Egypt). Follicles were classified according to the criteria listed in the Nomina Histologica into the following groups: secondary, tertiary, atretic, and cystic follicles (Nomina Histologica, 1994).

 The secondary antibody goat polyclonal anti-mouse and anti-rabbit IgG was purchased from Chemicon Dako (Life Trade, Cairo, Egypt). Each primary antibody was assayed in at least 5 sections of each ovary from each individual. A streptavidin-biotin immunoperoxidase method was used as described by (Salvetti, 2004). Immunoassay for hormones

Sections were deparaffinized and hydrated, and then microwave pretreatment (antigen retrieval) was performed. Endogen peroxidase activity was inhibited with 1% H2O2, and nonspecific binding was blocked with 10% normal goat serum. All sections were incubated with primary antibodies for 18 hours at 4uC, and after being washed in phosphate-buffered saline, the samples were incubated for 30 minutes at room temperature with preabsorbed biotinylated secondary antibodies selected specifically for each of the 2 types of primary antibodies used (monoclonal or polyclonal). The visualization of antigens was achieved by the streptavidinperoxidase method (BioGenex, San Ramon, CA, USA), and 3.3-diaminobenzidine (Liquid DAB-Plus Substrate Kit—Zymed, San Francisco, CA, USA) was used as chromogen. Finally, the slides were washed in distilled water and counterstained with Mayer’s hematoxylin, dehydrated, and mounted. Positive control tissues were used. Negative control sections were subjected to the same immunohistochemical method, replacing primary antibodies by rabbit and mouse nonimmune serum.

The ovarian follicles were classified according to the criteria listed in the Nomina Histologica into the following groups: primary, secondary, tertiary, atretic, and cystic follicles.

Clinical examnation of she camels and sampling:

Three reproductively sound, non pregnant, non-lactating female dromedary camels belong to Veterinary Teaching Hospital of the Faculty of Veterinary Medicine, Assiut University, under natural day light and local environmental condition were used in the present study. Animals aged 8-12 years and weighed approximately 450-500 kg. All animals were maintained in good body condition with suitable hygienic measures and they tied and kept outdoors under shelter. She-camels in the present study were subjected to daily examination using ultrasonography to monitor the changes in the size of different follicles especiallly dominant follicles throughout a complete follicular wave. All scans were performed at a pulse repetition frequency of 7.5 Hz. Images were used to calculate the diameter of each follicle during examination. According to the size of follicles, they were categorized into three classes; small (less than 10 mm), medium (10-15 mm) and large (more than 15 mm). According to the size of follicles, they were categorized into three classes; small (less than 10 mm), medium (10-15 mm) and large (more than 15 mm).

Blood serum and follicular fluid samples were analyzed for progesterone, estrogen, through EIA technique, using a Microstrip Elisa Reader (Stat-Fax-303, Awareness Technology, Inc.). Progesterone and estradiol concentrations were determined by using kits from Bremancos Diagnostic INC–GmbH, Germany (Cat. # BC-1113 & BC-1111, respectively). The lowest detectable level of progesterone during this test was 0.05 ng/ml, while the cross reactivity with other steroid hormones was <0.74%. For estradiol, the lowest detectable level was 5.9 pg/ml and cross reactivity with other steroids was <2.10%.

Serum (before slaughter) and Follicular fluid was obtained from 10 she camels slaughtered at Cairo central abbatoir and classified according to their size in the same order as in live animals using ultrasonography. Five pregnant She camels were sacrificed during their first trimester of pregnancy (2-4 months) were used to estimate progesterone in serum (before slaughter) and CL and relate them to E/P receptors. Serum was separated from the blood samples Coles (1986). CL and eppendorf tubes containing serum and follicular fluid were wrapped separately in aluminum foils and kept at −20 °C till analysis. Analysis was performed within 2 weeks from collection. Corpus luteum was homogenized at 1,500 rpm for 10 min in phosphate buffer, pH 7.2, for extraction of the homogenized samples; 1 ml ethanol containing butylated hydroxy toluene and 3 ml hexane were added to 1 ml homogenized tissue. The mixture was shaken vigorously for 10 min by hand and centrifuged at 800×g for10 min.

Progesterone and Estradiol assay

Blood samples were collected daily from the jugular vein from she camels throughout the studied period and centrifuged immediately after collection at 1700 xg. The harvested plasma was stored at -20 ºC until hormonal analysis. Progesterone concentration was determined with a commercially available Progesterone EIA kit (FERTIGNIX-PROG-EASIA). The range of the standards used was 0–11.5 ng mL_1. The inter- and intra-run precision had a coefficient of variation of 2.9 and 4.8%, respectively. Plasma concentrations of estradiol-17β (E2) was determined by ELISA using commercial kits (Human Gesellschaft fur Biochemica und Diagnostica, Wiesbaden – Germany). The coefficient of variance of intra- and interassay were 5.2 and 9.3%, and the sensitivity of the assay was 3 pgml−1.

Statistical analysis

Statistical analysis:

Statistical analysis of the collected data was carried out according to procedures of completely random design, SAS (1995).

The data of hormonal concentration were follicular sectional diameter (SD were expressed as mean ± SEM. When main effect of group or group by day was observed, the difference of group means at specific time point were analyzed by the Student’s t-test using JMP statistical software (version 5.1; SAS Institute, Cary, NC, USA). The different means were significant at P<0.05.

RESULTS

Morphologically, the ovary of the she-camel was flattened, lobulated measuring 3.17, 2.21 and 0.8 cm length, width and thickness respectively. Both right and left ovaries exhibited follicles in various stages of development, including primordial, primary, secondary and tertiary follicles, corpora albicantia, and late CL, as well as follicles with different degrees of atresia. The measurements of the different follicles and corpora lutaea during different stage of the estrus cycle were summarized in (Ttable. 1) and the histological morphmetric measurements of the oocytes in different types of the ovarian follicles.

(tab. 2)

Immunohistochemically: In all examined she- camels, immunoreactivity of ERα  was observed detected iatn low  amounts in the follicular cells of the primordial and primary secondary follicles, moderate corpora lutea and corpora albicantia. hMoreover, it was recorded at high amount in the vital and atretic tertiary follicles. , corpora lutea and corpora albicantia. Also, ERα was detected in cells of the deep and superficial stroma, tunica albuginea and surface epithelium but the reaction was weak in stroma cells surrounding the follicles. The reaction was strong in the cytoplasm of the ova of the primary and secondry follicles but was absent in the granulose cells surrounding these follicles (Fig.1-&2). Furthermore, immunostaining of ERα was detected in cells of the deep and superficial stroma, tunica albuginea and surface epithelium (Fig. 2 & 3).

In mature ovarian follicles (Fig. 4); ERα was strongly expressed in the cellular nuclei and cytoplasm of the granulose, and moderate in that of theca interna, and theca externa layers. from all follicular categories studied. Granulosa cells were strongly stained with 1D5 clone. The reaction products in this cell type were granular in the nucleiand in the cytoplasmproducts in this cell type were granular in the nuclei and in the cytoplasm of the granulose cells but were . homogeneous distribution in theca interna and theca externa. In corpora lutea, scoresERα was detected in low amount for both large and smallin the lutein cells and  were low, whereas in the stroma cells. the score was still lower. Moreover, itIn corpora albicantia the ERαimmunostaining was strongerhigher in the capsular stroma than in the internal stroma.

ERα was widely dispersed in the theca interna and theca externa, it showed moderate staining intensity with homogeneous distribution in the cellular nucleus in theca interna and strong staining in the theca externa.

ERα was not detected in cells of the corpora hemorrhagica..

Localization of PR in the camel  ovary

On other hand, In all samples , immunostaining of PR was detected as moderate reaction in cells of the stroma, corpora lutea and corpora albicantia, tunica albuginea and stroma cells surrounding the follicles surface epithelium but the reaction wasis strong in the blood vessels (Fig. 5 ). In addition, PR was But observed detected in low amounts in secondary follicles, vital and atretic  and tertiary follicles,. Furthermore in mature ovarian follicles, PR was expressed weak reaction in the cellular cytoplasm and nuclei of the granulose cells. The immunostaining was low or absent in the theca externa cells and strong in the cytoplasm and nuclei in the superficial layer of the mature follicles.

Estrogen concentration (Table 1 and Histogram2)  in both the follicular fluid (r=-0.06) and serum (r=-0.15) correlated negatively -but not significantly- with the size of the follicle while a positive non significant correlation was found between serum progesterone and the size of the corpus luteum. However, positive non significant correlation was detected between serum progesterone and the size of the corpus luteum in the studied animals throughout the days of the cycle (r=0.25). 

Serum and follicular fluid estrogen (Table. 2& 3le 1 and Hist. ogram21& 2 ) was higher in follicles exceeding 15 mm more than the lesser follicular categories. Slight difference in the concetration of estrogen was found between follicles less than 10 mm diameter and those between 10 – 15 mm. A highly positive correlation (Table.le 12 and Hist. stogram32) was detected between the size category of the corpus luteum and serum progesterone concentration (r= 0.99, p<0.0001).

(Tab. 1): The measurements of the ovarian follicles and the serum levels of the steroid hormones during the different stage of the estrus cycles

Table 1: The morphmetric measurements of the oocytes in µm in the different ovarian follicles measured from histological sections of the she-camel ovaries stained with H&E

Table 2: Estrogen (E₂) concentration in follicular fluid and serum of different size classes of ovarian follicles of she camels.

Table 3: Progesterone concentration in she camels relative to the size of the CL

Hist. (1): Estrogen (E2) concentration in follicular fluid and serum of different size classes of ovarian follicles of she camels.

Hist. (2): progesterone concentration in she camels relative to the size of the CL

1      2 

Fig. 1- 4: Micrographs of different she-camel ovarian cell types at various estrous stages showing immunostaining of ERα. By Streptavidin-Biotin method, Mayer's hematoxylin counterstain.

1- Outer region of the ovarian cortex during estrus with high immunoreactivity for ERα (SERα) in the surface epithelium, low immunoreactivty in the superficial stroma, and strong expression in the tunica albuginea, cytoplasm and nuclus of  oocytes of the primordial follicles.X200.

2- Primary follicle of she-camel ovary showed ERα strong immunoreactions in the cytoplasm of oocyte and moderate reaction in the follicular cells. X100.

3- Low ERα immunostaining in the wall of a secondary follicle (s), stroma cells and atric follicles during proestrus. X100 .

4- Wall of the mature ovarian follicle presented strong immunoreactivity in the cytoplasm and nuclei granulosa, whereas theca interna and externa show moderate reaction but few nuclei show strong reaction. X200.

Fig. 5-6: Micrographs of different she-camel ovarian cell types showing immunostaining of PR. By Streptavidin-Biotin method, Mayer's hematoxylin counterstain.

5- PR immunoreactivity was moderated in corpus luteum, cells of the stroma and blood vessels. X40.

6- In the mature ovarian follicle, the PR immunoreactivity was low in the theca interna and externa but high in granulose layer X40

DISCUSSION

The obtained immunohistochemical observations indicate that the expression of ER and PR and the secretion of their specific hormones in the ovary of Arabian she-camel were well correlated with the reproductive cycle. But during ovarian activity, the expression of ER and PR is not always correlated with the presence of the hormones in the follicles and serum. The immunohistochemical expression of the ER was detected in the nuculei and cytoplasm of the ovarian cells whereas in the primate ovaries, the ER of the granulosa cells was nuclear only (Billiar et al., 1992; Suzuki et al., 1994 and Saunders et al., 2000). In rat, hamster and pig, the ER is nuclear and cytosolic, and the cytosolic fraction is more important (Kawashima and Greenwald 1993). According to Guiochon-Mantel and Milgrom (1993), ER is essentially localized in the nucleus in the absence of estrogens. In addition, at the hypothalamic and hypophysal level, the estradiol injection induces the increase of the cytosolic fraction of the ER (Kawashima et al., 1987). In she-camel ovary, the variation during the reproductive cycle would translate a functional action.  Numerous studies show on the contrary that the theca cells are the major sites of ER expression in different speciesincluding rats, mice and primate (Chandrasekher  et al., 1994; Kuiper, et al., 1996; Sanchez-Criado et al., 2005) or total absence of the ER in the ovary (Saunders et al., 1997).

As reported in the bovine ovaries (Van den Broeck et al., 2002), in the present study, the PR localized in the different ovarian structure of the she-camel additionally shows that the various ovarian cell types exhibit different patterns of PR immunoreactivity during the ovarian activities. In the follicle cells of primordial, primary and secondary follicles the scores for PR were high and increased from primordial to secondary follicles. These data are in accordance with findings in primates (Hild-Petito et al., 1988) and dogs (Vermeirsch et al., 2001), and they indicate that progesterone may regulate follicular growth during the early stages of follicular development.

In she-camel follicular structures examined, the follicle/granulosa cells showed the high PR immunostaining during oestrus, when ovulation occurs. These results are concomitant with earlier observations in the dog (Vermeirsch et al., 2001) and with a study on PR mRNA in the bovine ovary (Cassar et al., 2002). The crucial role of PR in the ovulatory process has been demonstrated in PR-deficient mice, since such mice develop large follicles but fail to ovulate (Lydon et al., 1995). All these findings emphasize the important role of progesterone and its receptor in the ovulation process. The expression of PR in granulosa cells in tertiary follicles is induced by the LH surge (Hild-Petito et al., 1988). The induction of PR mRNA has also been observed in monkey granulosa cells during periovulatory stages (Chandrasekher et al., 1994) and in porcine granulosa cells cultured in vitro after LH stimulation (Iwai et al., 1991). The progesterone receptors are reported to mediate the protective effects of progesterone against apoptosis in the granulosa cells of preovulatory follicles (Quirk et al., 2004).

The presence of PR in corpora lutea reflects the role of progesterone in corpus luteum activity (Revelli et al., 1996; Rueda et al., 2000). Progesterone regulates the proliferation and development of luteinized granulosa and theca cells in an autocrine and paracrine way (Sasano and Suzuki, 1997). The presence of PR in all lutein cells of the corpora lutea suggests the influence of progesterone in the luteinization process (Revelli et al., 1996; Duffy et al., 1997). However, in the present study, the PR immunostaining in the corpus luteum was lower than in most other ovarian structures, which can be due to a negative effect of the locally produced high levels of progesterone to the PR production. In contrast to all other ovarian cells, the lutein cells of the corpora lutea showed PR immunostaining not only in the nuclei, but in the cytoplasm as well.

A low but manifest PR immunoreactivity was observed in cells of the tunica albuginea and the surface epithelium. This corresponds with a study on ovine ovaries in which it has been suggested that cells of the ovarian surface epithelium are enzymatically involved in the ovulation process by the influence of progesterone and its receptors (Murdoch, 1998). Further investigations in cattle are necessary to verify the role of PR and progesterone in the ovarian surface epithelium.

The present study indicated that estrogen concentration in both the follicular fluid (r = 0.06) and serum (r = 0.15) correlated negatively (non-significantly) with the size of the follicle while a positive non significant correlation was found between serum progesterone and the size of the corpus luteum. However, positive non significant correlation was detected between serum progesterone and the size of the corpus luteum in the studied animals throughout the days of the cycle (r = 0.25). On other hand, present immunohistochemical observations indicate that the expression of ER and PR and the secretion of their specific hormones in the ovary of camel was not always correlated with the presence of the hormones.

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