The consistent repetitive estrous cycle, which last for 18- 21 days in bovine, have many stages, which are governs by different developmental and hormonal changes (Garcia et al. (2002). The estrous cycle starts on day 0, and terminated with the lysis of the corpus luteum on day 18-21 Northey and French (1980). On day 0 the follicle ruptures, freeing the egg to the in findulum Johnson (2015). Instantly after ovulation new types of cell termed as luteal cells develop inside the ovary from the granulosa cells, rather promptly up to 5-6 days these cells rise to a mature corpus luteum (Koering et al. 1964). Even though the term corpus luteum was lead to Marcello Malpighi in a communication to Jacobo Spon, the major explanation and sketches of corpus luteum was completed by Catchpole (1940) who advised globular bodies in ovaries of pregnant rabbits (Jocelyn and Setchell, 1972). Corpus luteum, a temporary endocrine body which secretes progesterone (P4), which is required for pregnancy maintenance and establishment (Devoto et al. 2017). In the lack of pregnancy, the CL regressed by its own called luteolysis. This process is well defined as the supervision of gonadotropin releasing hormones (GnRH) adversaries origins a quick drop in the production of progesterone., although in cattle and rodents this influence is fewer affected (Niswender et al. 2000). The CL development, upkeep and deterioration are distributed into four stages I, II, III, and IV (Ireland et al. 1979). Regression and maintenance of corpus luteum is determined by complex interrelated events of the cell cycle (Bachelot and Binart, 2005). The cell cycle of mammals, defined as a series of procedures among two cells division is surely regulated by cyclin and cyclin dependent kinases (CDKs), which acquaintance to make heterodimeric facilities (Sherr, 1996) and adversely controlled by CDKIs i.e. p21 and p27 (Taguchi et al. 2004). CDKIs are significant adverse controllers of cell cycles headways. Once tie CDK-cyclin complexes in the cell cycles’ G1 phase (Cell cycle), they chunk the CDKs activities, inhibiting phosphorylation of members of the Rb genes family and travel to S phase (Deng et al. 1995). Here we have two groups or family of physically different CDKIs. The CIP/KIP family that hinder a wide-ranging CDK’s by selectively tie and preventing the fully linked cyclin-CDKs complexes. The second family is the inhibitors of kinases (INK) family, that bind precisely to CDK4 or CDK6 and hinder composite development with cyclin D (Taguchi et al. 2004). The p21 and p27 have its place in the CIP/KIP family of Cdk’s inhibitors; both have effects on numerous CDKs in vitro (Besson et al. 2008). The cyclin E (protein) and CDK2 complexes can thus be deactivated by attachment of p21 and p27, hat result the arrest of G1 phase resulting absence of Rb phosphorylation (Deng et al. 1995). Due to high degree of similarities in protein initial structure, both the p27 and p21 are considered to be hindering their goals by comparable machineries. Grounded on morphological study (Russo et al. 1996), it is now believed an alpha-helix of a CIP/KIP protein pledges its first interaction with cyclin Aparicio et al. (2014). Importantly CDK2 conformational variations furthermore and bolt the catalytic forked in an indolent method. Astonishingly, the massive bulk of cyclin-D and CDK’s complex cover p27 or p21 (Sherr and Roberts, 1999). The CL undergoes both progression and regression while p21 and p27 genes are mainly involved in these processes, so it is imperative to determine the role of these genes in CL. Briefly the expression of both genes in corpus luteum have been determined. As previous studies have shown, the proven role of p21 and the p27 proteins as anti-apoptotic, anti-cancerous and anti-proliferative genes (Murad et al. 2016). This study will contribute to the detect the health status of pregnancy with CL development as well as the impact of expression level of the genes.
MATERIALS AND METHODS
Indigenous cattle (n=40) were randomly selected from a local abattoir situated at Kohat road Peshawar, Pakistan. Ovaries samples were collected from all those slaughtered animals via surgical tools. Those ovaries were transported to the lab in ice box. Later on corpus luteum were gently removed from the ovary and were kept for further biological processes (Kiyma et al. 2016). 12 samples were excluded in the later stages.
Determination stages of CL
The stages were determined on the basis of morphology. Each stage has its own characters i.e. exterior and interior appearances, diameter, surface vasculature and follicles defined by (Ireland et al. 1979).
Processing of CL
The excised corpus luteum was cut into two pieces, each part was about 5mm. One part was used for biological assessments i.e., for RNA extraction, while the second part was stored in 10% formalin for histopathology. For RNA extraction, the samples were minced or grinded in liquid nitrogen. Instantly transferred into a 2ml Eppendorf tube with 1ml TRiZol reagents for instant processing or was stored in RNA later@ solution for later processing.
Briefly, RNA were extracted from all 28 samples by TRiZol method through a manufacture’s protocol (Chomczynski and Sacchi, 1987). The process was performed inside late fetal heart (LFH) because of the RNA sensitivity. RNA concentrations (standard: 1.7 to 1.9) were checked through Nano Drop and was diluted to obtain uniformity (Figure 1).
The cDNA synthesis
The cDNA synthesis procedure is also described by Atli et al. (2010). Meanwhile 2 μg of the total RNA was reverse transcribed into cDNA through RevertAid cDNA synthesis Kit (Thermo Fisher Scientific CN: K1622 through reverse transcription PCR).
Figure 1 Extracted RNA gel electrophoresis
Amplification of the desired fragment
PCR was used to check the expression profile of p21 and p27. The procedure has defined as follow: 1 μL of the cDNA was taken with 10 μL of PCR Master Mix. 5 picoMOL of each primer were added, finally, ddWater were added to final volume (20 μL). Primers were obtained from a literature and blasted in NCBI for confirmation (Table 1). The GAPDH gene was spotted to be the best steady housekeeping gene, was hired to standardize the PCR figures of the studied p21 and p27 (Kiyma et al. 2016).
To verify the reaction specificity, each sample was separately run on 1% agarose gel. The amplification products were stained with ethidium bromide. 100 bp ladder was used for size comparison. The gel bands were captured through gel-doc (Figure 2).
The samples were first dehydrated with different concentration of alcohol (50, 70, 90 and 100%). After dehydration the samples were cleaned up with xylene. Samples were then cut into slices with the help of microtome, embedded with paraffin wax. The samples staining was done with H&E stain. After staining samples were fixed in buffer formalin. Slides were made and were examined with the help of various magnifications (4X, 10X and 40X).
The data thus obtained were subjected to one-way ANOVA (SPSS, 2011). The bands intensity was checked by ImageJ and the figures were made through PRISM graph-pad.
RESULTS AND DISCUSSION
Activity of p21 during different stages of the corpus luteum
In this study we checked the expression of p21 in different stages of the corpus luteum. The relative expression of p21 was significantly (P≤0.05) higher in stage II and III as compare to stage I and IV as shown in the Figure 3. During stage II and III, the activity of the luteal cells was higher for the production of progesterone, the expression of the p21 help in the protective mechanism of these luteal cells. During the regression stage IV and initial stage. I of luteal cells differentiation the activity of the p21 was minimal, showing that anti-apoptotic activity of the p21 during progression of the corpus luteum.
Relative expression of p27 in four stages of corpus luteum
The Figure 4 shows average expressions of p27 gene in four stages of CL. The overall results show that p27 expression is significantly lower in stage I but increased gradually up to stage-II and-III, while a quick decline occurred in stage-IV. These results further strengthened our hypothesis that p27 has a crucial role in CL maintenance mainly and regression as well. The results show that during progression stage of high activity of the luteal cells the higher expression of the p27 effect the apoptosis of the luteal cells to sustain its activity.
Proliferation of the luteal cells during different stages of CL
Figure 5 shows the proliferation of luteal cells during different stages of the corpus luteum. During stage I the cells started differentiation into the immature luteal cells followed by nuclear and cytoplasm division as shown in Figure 5a. The stage-II and -III show higher number and proliferation of the luteal cells, surface vasculature and large sized vesicles with appearance of some immune cells as shown in Figures 5b and 5c. During stage IV when the expression of p21 and p27 become lower, the cells collapsed and the apoptosis was increased while proliferation of the cells decreased which supported the anti-apoptotic activity of the p21 and p27 (Figure 5d). To confirm the role of p27 in the regression of CL, a supportive statement was reported by Katayose et al. (1997). They demonstrated that high levels of p27 expression induce apoptosis, although the effects of p27 in promoting cell death may be more indirect. The expression of p27 is very high in the stage-IV of CL as shown in Figures 3 and 4. These results indicate that p27 lower expression induced luteolysis in cattle.
Figure 2 P27 PCR products gel picture
Table 1 Primers21 and p27 NCBI results
Figure 3 Relative expression of p21 in different stages of corpus luteum
Figure 4 Relative expression of p27 in different stages of corpus luteum
The expression of p27 is lower in CL as compared to p21 because p27 is expressed at high levels in lymphocytes and in normal non-proliferating mammary epithelial cells serving as internal control cells in the immunohistochemically evaluation of p27 expression (Porter et al. 1997). Our results are in the line with Ghanem and Steinman (2005). Interestingly, p21 has conflicting roles in apoptosis, having been demonstrated to both promote and inhibit programmed cell death. The pro-apoptotic activity of p21 has been attributed to both p53-dependent and p53-independent regulation of the apoptotic effector protein bax. Their hypothesis clearly suggested that p21 and p27 has dual role in nature like they are apoptotic as well as anti-apoptotic. This nature can be distinguished on the basis of their expression level. Dramatic changes in expression of two cell cycle regulators, p21, p27 occur during the first 48 h of luteinization in vivo (Robker and Richards, 1998). The data of Robker and Richards (1998) and Richards et al. (1998) and our results show that p27 accumulates early in the development of luteinized tissue. It is shown here that p27 is maintained at this high level until down-regulation that occurs after about day 18 of pregnancy. Moreover, changes in cyclin D3, CDK4, and CDK6, and in their associations with p27, occur with dynamics similar to that of p27. Taken together, fully luteinized cells are devoid of any major fluctuations in cell cycle regulatory machinery until all the molecules studied here change together late in luteal life (Richards et al. 1998).
Figure 5 Histopathology of corpus luteum
a) A: follicle secreted area; B: theca cells; C: nuclear division; D: large vesicle; E: fresh cells injury and F: proliferation of luteal cells
b) A: surface vasculature; B: proliferation; C: vesicles; D: Immune cells and E: typical nuclei
c) A: connective cells proliferation; B: theca cells; C: cell collapse; D: regressed cells; E: vesicles and F: apoptosis
d) A: suppressed granulosa cells; B: cell structure destroyed the vasculature; C: apoptosis; D: chromatin condensation; E: unequal nuclear and cytoplasmic ratio and F: tissue and vesicles enlargement
Considering the expression level of p21 and p27 gene, the results showed a dynamic pattern in four stages of CL, as p21 expression in stage I and-IV is significantly lower while it higher in both stage-III and-IV. These results showed that the role of p21 expression in the luteinization and regression is lower than the maintenance of CL. The difference in the expression level of p21 and p27 gene in four stages may be due to the fact that experimental animals might have age differences or probably their genetic makeup might be different. Or some of the animals could be pregnant. On the other hand as p21 in an anti-apoptotic gene as well Gomez-Manzano et al. (1997), so it could have higher expression in the regression of CL but these results showed a slight contradiction with the dual nature of p21 as an apoptosis and anti-apoptosis (Ghanem and Steinman, 2005).
Overall, this study showed the expression pattern of p21 and p27 genes in different stages of corpus luteum. The lower expression in initial stages indicated the higher level of maintenance and recruitment, while the higher expression of both genes was found in the regression stages of corpus luteum. The results and comparative analysis showed that their functions maybe significantly related to apoptosis.
This research was financially supported by HEC, under project title “unrevealing the action of interferon-tau in the development and maintenance of the bovine corpus luteum”. I also wish to show my gratitude to Institute of Biotechnology and Genetic Engineering, the University of Agriculture Peshawar because its assistance was a milestone in the completion of this project.