Proliferating Cell Nuclear Antigen (PCNA) Localization in the Ovaries of Buffalo, Dog and Pig by Immunohistochemistry

Proliferating cell nuclear antigen (PCNA) is a ring-shaped homotrimer that encircles the DNA (Boehm et al., 2016; Dieckman et al., 2012). It was a proven fact that PCNA is a marker for cellular proliferation (Strzalka and Ziemienowicz, 2011; Strzalka et al., 2015) and it orchestrated the events necessary for DNA synthesis, repair, damage avoidance, cell cycle control and cell survival (Prakash et al., 2005; Helleday et al., 2007; Mirkin and Mirkin, 2007; Stoimenov and Helleday; 2009; Sun et al., 2012; Yin et al., 2015). It is an ideal marker of cell proliferation in both non neoplastic and neoplastic cells. Because of its essential role in promoting proliferation, PCNA is an obvious target for cancer therapy (Wang, 2014).

PCNA was used to demonstrate the ovarian follicle counts in different laboratory and farm animal species (Muskhelishvili et al., 2002; Phoophitphong et al., 2012, Machado et al., 2018). Immunohistochemical localization of follicles and oocytes with proliferating cell nuclear antigen (PCNA) was reported in ovaries of rat (Oktay et al., 1995, Picut et al., 2008), mouse (Kerr et al., 2006), rabbit (Hutt et al., 2006), pig (Tomanek and Chronowska, 2006) and sheep (Patel et al., 2018). To our knowledge, there was no detailed study of proliferation process throughout the follicular development in buffalo and dog ovaries. Hence, the present study was undertaken with an objective of immunostaining of ovaries with PCNA in order to identify the distinct pattern of follicular cell proliferation in different follicular stages. The pattern of PCNA staining in the ovary was compared to previous studies in pig ovary.

 

The following immunohistochemistry work was conducted in the Department of Pathology, Padmavathi Medical College, SVIMS, Tirupati during 2018-19.
 
Sample collection and tissue handling
 
The ovaries of buffalo, pig was obtained from a local abattoir and those of dog were collected from the dogs underwent ovariohysterectomy as a part of animal birth control programme in Veterinary Clinical Complex, College of Veterinary Science, SVVU, Tirupati during 2021. The ovaries were transported to laboratory in a thermo-container filled with PBS at 37°C. Ovarian tissue was cut into small pieces and fixed in 10% neutral buffered formalin solution. After fixation, the specimens were dehydrated by ascending grades of ethyl alcohol, followed by clearing with xylene and they were embedded in paraffin (Merck, Germany). The pieces of ovarian tissue were serially sectioned at 5 μm with the help of microtome (Leica RMZ 2125) and mounted on APES (Amino Propyl Triethoxy Silane) coated slides and incubated overnight at 37°C. These slides were subjected to the following immunohistochemistry protocol (Luna, 1968; Lynch, 1969).
 
Immunohistochemistry
 
Prior to PCNA localization, sections were deparaffinised by two changes of xylene 15 minutes each and rehydrated in decreasing concentrations of ethanol and PBS. The slides were kept in Tris EDTA buffer (pH-9.0) and subjected to heat induced epitope retrieval (HIER) treatment for 20 minutes at 100°C in a water bath to retrieve the antigenic sites and then cooled to the room temperature. The slides were kept in the distilled water for 5 minutes and in Tris buffer saline for 5 minutes. In order to block the endogenous peroxidase the slides were kept in the peroxidase block solution (3% hydrogen peroxide in methanol) for 10 minutes. Then slides were washed in Tris buffer saline for five minutes each in three changes. The power block solution using 1.5% bovine serum albumin (BSA) was poured on tissue section and kept for 30 minutes. Monoclonal mouse anti-PCNA (DAKO, Carpentaria, CA, USA: 1:200 dilution) was added on the sections and slides were kept at 4°C overnight. The next day, sections were washed in Tris buffer saline for five minutes each in three changes. The secondary antibody with horse radish peroxidase (HRP) was added and kept for 30 minutes at room temperature. Then sections were washed in Tris buffer saline for five minutes each in three changes. The binding of primary antibody was visualized using diaminobenzidine (DAB, Sigma, Germany) for 5-8 min. After washing with distilled water, sections were counterstained with Harris haematoxylin for 1 minute. The slides were washed in tap water for 5 minutes. For negative control, PBS was used instead of primary antibody. Then air dried and mounted with DPX. The PCNA labelling was examined using Leica DMLB microscope and the images were recorded by Leica DC 200 digital camera.

Follicles were classified according to the stage of development, considering the shape, and layers of the granulosa cells (GC) as follows: primordial (oocyte surrounded by a flat pre granulosa cell layer), primary (one layer of cuboidal granulosa cells), secondary (two or more layers of cuboidal cells but without formation of vesicles), early antral (three or more layers of cuboidal granulosa cells and the presence of antrum) and large antral follicles (Da Silva Buttkus et al., 2008). In the present study the intensity of reactivity was categorised as mild, moderate and strong.

The immunostaining with PCNA was multifactorial i.e., depending on the number of proliferating cells in the tissue sample, the concentration of primary antibodies, the pre-treatment of tissue with heat-induced epitope retrieval (HIER) technique, the type of fixative used (Muskhelishvili et al., 2005) and the time of fixation of the tissue (Takahashi et al., 1993).

The primordial follicles appeared as clusters in the connective tissue beneath the surface epithelium in case of pig, where as in most of the buffalo and dog ovaries they appeared mostly distributed in connective tissue as single follicles. The nucleus of ovum showed strong positive reactivity with PCNA (Fig 1, 2 and 3). Even though the ovum of primordial follicle was quiescent in diplotene stage of meiosis I (Byskov, 1982), it was possible that DNA polymerases were activated to repair potential damage to the genetic material in the oocytes selected to grow (Oktay et al., 1995). 



The flat pregranulosa cells did not show immunoreactivity in all the three species under study (Fig 1, 2 and 3). These reports in pig were contrary with Phoophitphong et al., (2012) as they observed immunoreactivity in pregranulosa cells in addition to that of ovum. No immunostaining for PCNA in the primordial follicles (either in oocyte or in pregranulosa) was reported earlier in bovine (Wandji et al., 1996), baboon (Wandji et al., 1997) pig (Tomanek and Chronowska, 2006) and mouse (Rodrigues et al., 2009). Phoophitphong et al., (2012) standardised the technique for PCNA immunohistochemistry in pig ovaries by including antigen retrieval and using a dilution of 1:200 of mouse monoclonal anti-PCNA.

As the follicular development progresses the granulosa cells become cuboidal.  The ovum of primary follicle in buffalo (Fig 1), dog (Fig 2) and pig showed strong immunorectivity. Only few cuboidal granulosa cells showed immunoreactivity in the primary and early secondary follicles in dog (Fig 2, 4, 5). One or two granulosa cells of the secondary follicles showed mild immunostaining in buffalo and pig (Fig 6).

The ovum of early antral follicles showed strong reactivity in all the species under study (Fig 7, 8, 9). All the granulosa cells of buffalo and pig showed moderate to strong reactivity (Fig 7, 9) whereas in dog few granulosa cells showed moderate immunoreactivity (Fig 8). The theca cells not showed immunostaining at this stage of follicular growth.

In antral follicles, the ovum showed moderate immunoreactivity in buffalo, dog and pig ovaries. The two different types of granulosa cells of large antral follicles displayed distinct patterns in that the cumulus granulosa cells were shown to divide more frequently than the mural granulosa cells (Hirshfield, 1986). Correspondingly, our studies with PCNA localization revealed more cumulus granulosa cells stained for PCNA as compared to the mural granulosa cells. The theca interna and its blood vessels showed moderate to strong immunostaining for PCNA in all the species under study (Fig 10, 12). These results were in accordance with previous studies that demonstrated a PCNA signal in different stages of follicular growth (Oktay et al., 1995; Tomanek and Chronowska, 2006).

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In degenerating follicles the granulosa and thecal cells were strongly immunoreactive in the initial stages of atresia. As the degeneration progress the immunostaining decreases and disappears (Fig 11, 13, 14 and 15). In all the species under study the intensity of PCNA immunostaining was negatively correlated to the stage of advance of atresia. We observed that the higher concentration of PCNA labelling in the healthy follicles than that of atretic follicles which is parallel to the findings of Feranil et al.  (2004 and 2005). In the medulla the blood vessel wall was strongly stained by PCNA.

We observed that in all the species under study the PCNA immunostaining intensity decreased with the stage of advance of atresia and also higher concentration of PCNA labelling in the healthy follicles than that of atretic follicles.

The authors thank S. Usha Nandini, Lab Technician, Department of Pathology, Padmavathi Medical College, SVIMS, Tirupati for her technical assistance during immunostaining procedure.

Authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.