Immunohistochemical localization of progesterone receptors alpha (PRA) in ovary of the pseudopregnant rabbit

Abstract Progesterone plays an important role in the reproductive function and follicular development in mammals. The aim of the present study was to examine the localization of progesterone receptor alpha (PRA) in ovary of pseudopregnant rabbit by immunohistochemical methods. Samples were collected from 14 h. to 18 days of pseudopregnancy. At the first stage of pseudopregnancy (14 h.), the rabbit ovary showed moderate immunostaining of PRA in the granulosa cells and theca interna cells of preovulatory follicle and in the stroma cells. At the middle stage of pseudopregnancy (3-7 days), the rabbit ovary showed strong immunostaining of PRA in ovarian surface epithelial cells, follicular cells of the primary follicle, granulosa cells and theca interna cells of the growing and antral follicles. Moderate immunoexpression of PRA were observed in the large lutein cells and endothelial cells of the corpus haemorrhagicum and corpus luteum and in the stroma cells. At the end of pseudopregnancy (18 days) strong PRA reactions were detected in the small lutein cells of the regressed corpus luteum. Moderate to strong PRA immuno-expression were observed in the proliferated theca interna cells of the atretic antral follicles. The atretic large lutein cells of the regressed corpus luteum showed negative immunostaining for PRA. This study showed that the PRA positive small lutein cells of the regressed corpus luteum and the PRA positive proliferated theca interna cells of the atretic antral follicles were transformed into PRA positive interstitial gland cells. In conclusion, the present study had described the distribution of PRA in the ovary of pseudopregnant rabbit, which is not discussed before in the available literature. It also gives more information about follicular dynamic, formation and origin of interstitial glands, mechanism of ovulation, formation and regression of the corpus luteum.


Introduction
Progesterone (P 4 ) is a steroid hormone that plays an important role in female reproductive activity in vertebrates such as regulation of follicular development, growth and differentiation of ovarian structures, ovulation and luteinization. It also participates in maintenance of pregnancy and breast development (Spencer and Bazer, 2002;Conneely et al., 2003;Anzaldua Arce et al., 2010). It is well known that the effects of P4 are mediated by its interaction with specific hormone-binding proteins called progesterone receptor (PR). Progesterone receptor is belonging to the nuclear receptor superfamily that encoded by single gene which consists mainly of two different isoforms; progesterone receptors alpha (PRA) and progesterone receptors beta (PRB) (Conneely et al., 2002, Kubota et al., 2016.The amount of these receptors is under hormonal control (Blauer et al., 2005;Anzaldua Arce et al., 2010). They up-regulated by estradiol and downregulated by progesterone (Kraus and Katzenellenbogen, 1993;Anzaldua Arce et al., 2010;Camacho-Arroyo et al., 2003). Immunohistochemical localization of PR has been described in reproductive organs of mouse and human (Teilmann et al., 2006), ovaries of monkey, human, bovine and rabbit (Hild-Petito et al., 1988;Revelli et al., 1996;D'Haeseleer et al., 2007;Abd-Elkareem, 2017a) respectively and uterus of rat and pseudopregnant rabbit (Hegele-Hartung et al., 1992;Kraus and Katzenellenbogen, 1993;Abd-Elkareem, 2017a).
In our previous study (Abd-Elkareem, 2017a) we demonstrated the localization of PRA in rabbit ovary during pregnancy up to 10 days after pregnancy. The distribution of PRA in the ovary of pseudopregnant rabbit has not been discussed before in the literatures. Therefore, we carried out this study to demonstrate the immunolocalization of PRA in the ovary of pseudopregnant rabbit at different periods starting from 14hr to 18 days of pseudopregnancy. In addition to characterize the different levels of PRA expression in rabbit ovaries from 14 h to 18 days.

Animals and tissue collection
Thirteen mature female New Zealand white rabbits (2.4 ± 0.06 Kg body weight and 4-5 months old) were housed in separate cages under 22-25 ºC room temperature and controlled light (12 h light/ dark cycle) conditions.
Rabbits were induced to ovulate by intramuscular injection of HCG (50-70 IU Choriomon, IBSA Institute Biochimique S.A., Lugano, Switzerland). The day of induction was considered as 0 days of pseudopregnancy.
The protocol used in this experiment was approved by the Committees of use and care of experimental animals of Faculty of Veterinary Medicine, Assiut University, Egypt. DOI: 10.21451/1984 Anim. Reprod., v.16, n.2, p.302-310, Apr./Jun. 2019

Sampling
Right and left ovaries were collected at 14 h, 3, 7 and 18-days post-induction of ovulation. For each experimental period, ovaries of two to three animals were dissected immediately after slaughtering, and then they fixed with Bouin's fluid.

Immunohistochemistry
The fixed samples were processed for paraffin embedding.
3-5µm sections were prepared from paraffin embedded samples, then they dewaxed in xylene and rehydrated in descending grades of alcohol. Sections were rinsed in PBS (pH 7.4) three times 5 min each. According to Abd-Elkareem (2017a), the slides were incubated successively in 3% hydrogen peroxide for 10 min at room temperature, PBS (pH 7.4four times 5 min each). For antigen retrieval, the slides were boiled in 10 mM sodium citrate buffer (pH6.0) 20 min followed by cooling for 20 min at room temperature then rinsed in PBS (pH 7.4 three times 1 min each time). Immunohistochemical detection of PRA was performed using PR (Clone SP2) and an Ultravision Detection System (Anti-Polyvalent, HRP/DAB; Thermo Fisher Scientific, USA). To inhibit the nonspecific background, sections were covered with Ultra V block for 5 min at room temperature. Sections were incubated with rabbit monoclonal antibody (1: 300) (Cat.#RM-9102S0, Thermo Fisher Scientific, USA) for 30 min at room temperature, then were washed with PBS at pH 7.4 (four times 5 min each). For detection of the primary antibody, sections were then incubated with a biotinylated anti-Rabbit antibody (1:300), Thermo Fisher Scientific, USA) for10 min at room temperature. Incubation was followed by three 5 min washes in PBS (pH 7.4) then the sections were incubated streptavidinperoxidase complex (Thermo Fisher Scientific, USA) for 10 min at room temperature. Four times washes 5 min in PBS. Peroxidase activity was visualized by 5-15 min incubation at room temperature in a solution consisting of one drop of DAB (diaminobenzidine) Plus chromogen to 2 ml of DAB Plus substrate. Sections were counterstained in Harris hematoxylin. After washing in distilled water, the sections were dehydrated and cover slipped with DPX. Negative controls were performed as the previous steps without adding the primary antibody. Sections were examined using an OLYMPUS BX51microscope and photographed with an OLYMPUSDP72 camera adapted to the microscope. The assessment of intensity of the immunostaining in the nucleus and cytoplasm was dependent on its color: dark brown to black (strong or intense), brown (moderate), light brown (weak) and no immunoreactivity (negative immunostaining).

At
14 h of pseudopregnancy, immunohistochemical localization of receptors alpha (PRA) in the rabbit ovary showed moderate nuclear and cytoplasmic immunostaining of PRA in ovarian surface epithelial cells ( Fig. 1A and C) and granulosa cells of secondary follicles (Fig. 1F). Weak to moderate reaction of PRA were observed in cytoplasm of oocytes of the primordial follicles ( Fig. 1 C), in the interstitial gland cells (Fig. 1A), in the stromal cells (Figs. 1C and D), granulosa cells and theca interna cells of preovulatory follicle (Fig. 1E). Negative immunostaining of PRA was observed in some interstitial gland cells (1D), in the nucleus and cytoplasm of oocyte of secondary follicles (1F). Ovulation stigma could be demonstrated at this stage of pseudopregnancy in rabbits. It was the thinnest area of the ovarian surface where the preovulatory follicle will burst through during ovulation. Stigma in rabbit contained weak to moderate PRA immunostaining in the ovarian surface epithelial cells, macrophage, neutrophils, fibroblasts, intermingled granulosa cells and theca interna cells and in the mitotic figure of endothelial cells.
At 3 days of pseudopregnancy, the rabbit ovary showed strong nuclear and cytoplasmic immunostaining of PRA in ovarian surface epithelial cells ( Figs Immunostaining of PRA in the rabbit ovary at 18 days of pseudopregnancy showed strong nuclear and cytoplasmic reactions in the ovarian surface epithelial cells (Fig. 4A) and in the small lutein cells of the regressed corpus luteum (Fig. 4F). Moderate to strong PRA immuno-expression were observed in the granulosa cells of the small antral follicle (Fig. 4 B), 304 endothelial cells of the blood vessels (Fig. 4E) and in the proliferated theca interna cells of the atretic antral follicles (Fig. 4E). Moderate reaction of PRA was observed in stromal cells (Fig. 4E) and in the granulosa cells and theca interna cells of the large antral follicle (Fig. 4C). Moderate PRA expression was observed in the oocyte of the large antral follicle (Fig. 4C). Weak PRA reaction was observed in the oocytes of the primordial follicle (Fig. 4A), small antral follicle (Fig.  4B). and in the interstitial gland cells (Figs. 4 B,D and E). Negative PRA immunostaining were observed in the large lutein cells of the regressed corpus luteum (Fig. 4 C, D and F). In this stage of pseudopregnancy the connective tissue of the regressed corpus luteum was proliferated to divide the regressed corpus luteum into complete separate lobules each of which contained regressed lutein tissue (Fig. 4D). The regressed lutein tissue was formed of atretic large lutein cells (PRA negative) and healthy small lutein cells (strong PRA immunostaining) which transformed into interstitial gland cells. This explained why the interstitial gland was formed of several lobules.
Ovulation stigma could be demonstrated at 14 h of pseudopregnancy in rabbits. It was the thinnest areas of the ovarian surface where the preovulatory follicles will burst through during ovulation. It showed weak to moderate PRA immunostaining in the ovarian surface epithelial cells, macrophages, neutrophils, fibroblasts, granulosa cells and theca interna cells. PRA is essential for ovulation because mice lacking PRA don't ovulate and are infertile (Gava et al., 2004;Mulac-Jericevic and Conneely, 2004). ADAMTS-1 (A disintegrin and metalloproteinase with thrombospondinlike motifs) and cathepsin L (a lysosomal cysteine protease) are two proteases induced in highest levels after LH stimulation in granulosa cells of preovulatory follicles in a PR-dependent manner (Robker et al., 2000), they are involved in degradation of the follicular wall. It was reported that the cells of the ovine ovarian surface epithelium are enzymatically involved in the ovulatory process by the influence of progesterone and its receptors (Murdoch, 1998). This indicated that the progesterone and its receptors (PRA) play a central role in the formation of ovarian stigma in pseudopregnant rabbit and this occurred by the action of immune cells (macrophages and neutrophils), connective tissue cells (fibroblasts) and ovarian cells (granulosa and theca interna cells). LH surge induces expression of PR by granulosa cells and withdrawal from the cell cycle. Progesterone by binding to progesterone receptor inhibits apoptosis. All these events promote resistance to apoptosis in the granulosa cells of bovine preovulatory follicles (Quirk et al., 2004).
At the middle stage of pseudopregnancy (3-7 days), the rabbit ovary showed strong immunostaining of PRA in ovarian surface epithelial cells, in follicular cells of the primary follicle and in the granulosa cells and theca interna cells of the growing and antral follicles. These data are in accordance with findings in pregnant and pseudopregnant rabbits (Korte and Isola, 1988;Iwai et al., 1991;Parillo et al., 2013;Lan et al., 2014;Abd-Elkareem, 2017a). Progesterone (P4) plays an important role in the intraovarian regulation of follicular growth and development (Revelli et al., 1996;D'Haeseleer et al., 2007). Progesterone promotes all stages of follicular development and directly suppresses the final large follicular stages only (Setty and Mills, 1987). The effects of P4 are mediated by its binding with specific progesterone receptor (PR). In bovine ovary, P4 regulate follicular development through the interaction of granulosa cells, theca cells, and stroma cells (D'Haeseleer et al., 2007). Granulosa cells, thecal/ stromal cells secrete P4 at different levels (Peluso, 2006) and they may have a different PRA immunoexpression pattern during pseudopregnancy. The expression of PR in granulosa cells was regulated by the activities of hCG (Iwai et al., 1991).
The expression of PRA in thecal cells indicated that thecal cells may mediate some actions of steroid hormones on the follicle cells in growing and antral follicles (Vermeirsch et al., 2001). As estradiol is the principle luteotropin in rabbits (Keyes and Armstrong, 1968) and only follicles make a significant amount of estrogen (Mills and Savard, 1973), follicular development and steroidogenic activity is essential for continuation of the corpus luteum in the pseudopregnant rabbit.
The present study showed moderate PRA immunostaining in the large lutein cells and endothelial cells of the corpora haemorrhagica and corpora lutea at the middle stage of pseudopregnancy. These results were agreeing with findings in human (Sasano and Suzuki, 1997;Vazquez et al., 1999;Maybin and Duncan, 2004), in monkey (Hild-Petito et al., 1988), in cattle (Rueda et al., 2000;D'Haeseleer et al., 2007) and in rabbit (Parillo et al., 2013;Abd-Elkareem, 2017a). Our results were disagreeing with findings in pseudopregnant rabbits (Korte and Isola, 1988) as in this study the corpora lutea of pseudopregnant rabbits contained small amounts of PRA. Progesterone receptors in the large lutein cells and endothelial cells of the corpora haemorrhagica and corpora lutea are required for luteinization, maintenance of luteal structure and function (Duffy et al., 1997) and they regulate vascularization of the corpus luteum (Vazquez et al., 1999). The main function of the corpus luteum is secretion of the hormone P4 which is required for maintenance of normal pregnancy via PRA. The stroma cells surrounding both follicles and corpora lutea were stained positive for PR (Revelli et al., 1996).
The present study revealed that at the end of pseudopregnancy (18 days), the regressed corpora lutea of the pseudopregnant rabbit showed strong PRA immunostaining in the small lutein cells while the apoptotic large lutein cells showed negative reactivity. This agree with our results in 10 days post parturient rabbit ovary (Abd-Elkareem, 2017a).
The present study showed that the PRA positive small lutein cells of the regressed corpus luteum and the PRA positive proliferated theca interna cells of the atretic antral follicles were transformed into PRA positive interstitial gland cells. PRA may play a role in this transformation (Abd-Elkareem, 2017a). Interstitial gland cells of the rabbit ovary had two main sources. The first one was the theca interna cells of the atretic antral follicles and the second source was small lutein cells of regressed corpora lutea which originated from the theca interna cells of postovulatory follicle (Abd-Elkareem, 2010; Abd-Elkareem, 2014). This