Abstract
The corpus luteum contains differentiated steroidogenic cells that have exited the cell cycle of proliferation. In some tissues, deletion of quiescent, differentiated cells by apoptosis in response to injury or pathology is preceded by reentry into the cell cycle. We tested whether luteal cells reenter the cell cycle during the physiological process of luteolysis. Ovaries were obtained after injection of cows with a luteolytic dose of prostaglandin F2α (PGF). In luteal sections, cells co-staining for markers of cell proliferation (MKI67) and apoptosis (cPARP1) increased 24 h after PGF, indicating that cells that reenter the cell cycle undergo apoptosis. The percent of steroidogenic cells (CYP11A1-positive) co-staining for MKI67 increased after PGF, while co-staining of non-steroidogenic cells did not change. Dispersed luteal cells were stained with Nile Red to distinguish lipid-rich steroidogenic cells from nonsteroidogenic cells and co-stained for DNA. Flow cytometry showed that the percent of steroidogenic cells progressing through the cell cycle and undergoing apoptosis increased after PGF. Culturing luteal cells induced reentry of steroidogenic cells into the cell cycle, providing a model to test the influence of the cell cycle on susceptibility to apoptosis. Blocking cells early in the cell cycle using inhibitors reduced cell death in response to treatment with the apoptosis-inducing protein, Fas ligand (FASL). Progesterone treatment reduced progression through the cell cycle and decreased FASL-induced apoptosis. In summary, steroidogenic cells reenter the cell cycle upon induction of luteal regression. While quiescent cells are resistant to apoptosis, entry into the cell cycle promotes susceptibility to apoptosis.
Introduction
Regression of the corpus luteum (CL) involves an initial loss of steroidogenic capacity, known as functional regression, followed by structural regression involving cell death by apoptosis and degeneration of the extracellular matrix (Juengel et al. 1993, Murdoch 1995, Carambula et al. 2002). In ruminants, the pulsatile release of prostaglandin F2α (PGF) from the uterus at the end of the nonpregnant cycle triggers luteolysis (McCracken et al. 1999). Events triggered by PGF in vivo are not mimicked in vitro, and it is likely that interactions among multiple cell types present within the CL and signals from a variety of pathways mediate PGF-induced luteolysis (Davis & Rueda 2002). The goal of the current study was to test the novel hypothesis that death of luteal cells during regression of the CL involves reentry of quiescent, differentiated cells into the cell cycle.
The CL is composed of a mixture of steroidogenic cells, endothelial cells, pericytes, fibroblasts, and immune cells (O'Shea et al. 1989, Niswender et al. 2000). Both granulosa cells and theca cells of ovulatory follicles differentiate to become steroidogenic cells of the CL. During the bovine estrous cycle, 60–70% of luteal cells represent nonsteroidogenic cells (Lei et al. 1991). Analysis of the cell proliferation marker, proliferating cell nuclear antigen (PCNA), in the early bovine CL (days 1–4) showed proliferative cells in areas presumed to be populated by theca-derived cells and few proliferating cells in areas presumed to represent granulosa-derived cells (O'Shea et al. 1989). During most of the bovine estrous cycle, the majority of proliferating cells were small cells including parenchymal cells, fibroblasts, and endothelial cells (O'Shea et al. 1989). This study and others (Farin et al. 1986) suggest that, in the ruminant, granulosa-derived luteal cells are predominantly nonproliferative during the estrous cycle, while theca-derived luteal cells are proliferative during the early luteal phase and become nonproliferative by the late luteal phase. Lack of proliferation of granulosa-derived luteal cells is consistent with studies on periovulatory follicles in cows (Quirk et al. 2004), rodents (Richards et al. 1998, Robker & Richards 1998), and macaques (Chaffin et al. 2001), which showed that the preovulatory LH surge induces the withdrawal of granulosa cells from the cell cycle.
There is increasing evidence for an interrelationship between cell proliferation and apoptosis. Cells proliferate by progression through a series of stages including G1 (first gap phase, before DNA synthesis), S (DNA synthesis phase), G2 (second gap phase, after DNA synthesis when cells prepare to undergo mitosis), and M (mitosis). Progression through the cell cycle is coordinated by a family of cyclin-dependent kinases (CDKs) that are sequentially activated by binding to cyclin regulatory proteins that are synthesized during specific portions of the cell cycle. Enzymatic activity of the cyclin/CDK complexes is regulated by phosphorylation of the catalytic subunits and by binding of CDK inhibitors such as cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21WAF1/CIP1) or CDKN1B (also known as p27KIP1), both of which interfere with the kinase activity of cyclin–CDK complexes. When cells terminally differentiate, they withdraw from the cell cycle of proliferation and reside in a resting, quiescent phase, G0. Experiments with a number of cell types have shown that withdrawal from the cell cycle is associated with development of resistance to apoptosis (Meikrantz & Schlegel 1995, Poluha et al. 1996, Wang & Walsh 1996, Schutte & Ramaekers 2000). In addition, our studies with bovine granulosa cells showed that their withdrawal from the cell cycle following the preovulatory LH surge is associated with increased resistance to apoptosis (Porter et al. 2001, Quirk et al. 2004). Furthermore, we found that culturing bovine granulosa cells with inhibitors that blocked cells in G0/G1 phase of the cycle promoted resistance to apoptosis (Quirk et al. 2004, 2006).
Many instances of neuronal cell death are associated with reentry of post-mitotic, differentiated cells into the cell cycle (reviewed in Klein & Ackerman (2003), Becker & Bonni (2004), Herrup et al. (2004) and Folch et al. (2011)). Pathological triggers of cell death in a number of other tissues are also associated with cell cycle reentry (Buttyan et al. 1988, Colombel et al. 1992, Pandey & Wang 1995, Wiesen & Werb 2000, Ecarnot-Laubriet et al. 2002). Because the fully developed CL is composed predominantly of differentiated, nonproliferating cells, it was of interest to determine whether the physiological process of luteal regression is associated with cell cycle reentry.
In the current study, cell cycle progression during luteal regression was assessed by histological staining for a cell proliferation marker, MKI67 (also known as Ki67), as well as by flow cytometry to assess the DNA content of cells and corresponding stage of the cell cycle. In addition, experiments with cultured luteal cells examined the relationship between stage of the cell cycle and susceptibility to apoptosis; progression through the cell cycle was manipulated by treatment with pharmacological inhibitors or by infection with adenoviruses expressing CDK inhibitors. In addition, effects on apoptosis and the cell cycle were examined in response to modulating signaling through progesterone receptor (PGR) or cellular production of nitric oxide. In these culture experiments, treatment with a soluble form of the transmembrane protein Fas ligand (TNF superfamily, member 6; FASL) was used as a tool to test susceptibility of cells to apoptosis. It is well established that FASL triggers apoptosis in sensitive cells upon binding to its cell surface receptor, FAS (TNF receptor superfamily member 6). The FAS pathway is present in most cells including luteal cells (Roughton et al. 1999, Quirk et al. 2000b, Taniguchi et al. 2002, Galvao et al. 2010, Atli et al. 2012), granulosa cells, and theca cells (Porter et al. 2000, Vickers et al. 2000).
Results
Immunohistochemical analyses of luteal cell types and expression of a cell proliferation marker
Injection of PGF to cows during the mid-luteal phase, on day 12 or 13 of the estrous cycle, caused a decrease in the concentration of plasma progesterone from 6.5±0.6 ng/ml at 0 h after PGF to 1.2±0.5 ng/ml at 24 h and 0.7±0.2 ng/ml at 48 h, indicating that induction of luteolysis occurred. Histological sections of CL were used to determine the percent of cells represented by steroidogenic cells, endothelial cells, and leukocytes and to determine whether the various cell types expressed a marker of cell proliferation, MKI67, and a marker of apoptosis, cleaved poly(ADP-ribose) polymerase 1 (cPARP1; Fig. 1A, B, C, and D). In CL sections co-stained for MKI67 and cPARP1, the percent of cells staining positively for MKI67 were relatively low in mid-cycle CL and increased between 0 and 24 h after PGF (Fig. 1A). The percent of cells staining for cPARP1 increased at 24 h, and this elevated level was maintained at 48 h (Fig. 1B). Examination of the co-staining pattern at 24 and 48 h showed that ∼20% of the cells that stained positively for MKI67 were also cPARP1 positive (Fig. 1C). By contrast, about 10% of the cells that were negative for MKI67 staining were cPARP1 positive (Fig. 1C). These results suggest that at least a fraction of cPARP1 positive, apoptotic cells observed in the CL after PGF had reentered the cell cycle. While relatively small differences in the percent of cells that are positive for MKI67 or cPARP1 are measured at single points in time, effects of cumulative changes in populations of cells over time could generate significant effects on luteal function.
Immunohistochemical expression of MKI67, a marker of cell proliferation, and cPARP1, a marker of apoptosis. The percent of cells expressing MKI67 (A) and cPARP1 (B) were compared 0, 24, and 48 h after PGF. The percent of cells expressing cPARP1 in cells that were MKI67-positive or -negative were also compared (C). Data represent the mean±s.e.m. of cell counts from four fields/CL from CL of three cows at each time. Within panels, bars without common superscripts are significantly different (P<0.05). (D) Representative fields of luteal tissue showing staining for MKI67 (green) and cPARP1 (red). Yellow cells are positive for both MKI67 and cPARP1. Nuclei are stained blue. Staining for MKI67 and cPARP1, which are nuclear antigens, are superimposed over nuclear counterstaining (i.e. not blended with nuclear counterstaining) in order to improve visibility. Scale bar=20 μm.
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0324
In order to determine the identity of proliferative cells within the CL, sections were co-stained for a marker of steroidogenic luteal cells, cytochrome P450 side chain cleavage (CYP11A1), and MKI67 (Fig. 2A, B, and C). CYP11A1 was selected as a marker because it was previously shown that CYP11A1 mRNA and protein show little or no decrease in expression in bovine luteal cells for at least 24 h after PGF, well after the decrease in progesterone (Tian et al. 1994, Rodgers et al. 1995). The percent of luteal cells staining positively for CYP11A1 decreased progressively at 24 and 48 h after PGF, consistent with the loss of steroidogenic cells during luteal regression (Fig. 2A). There was a significant increase in MKI67 staining among CYP11A1-positive cells at 24 h, followed by a return to lower levels at 48 h (Fig. 2B). This increase was not observed among CYP11A1-negative cells (Fig. 2B). Additional sections of CL were co-stained for the endothelial cell marker von Willebrand factor (VWF) and MKI67. There was a progressive increase in the percent of luteal cells that were positive for VWF between 0 and 48 h after PGF (Fig. 2D). The percent of VWF-positive endothelial cells that were also positive for MKI67 did not change after PGF, while VWF-negative cells had increased expression of MKI67 at 24 h (Fig. 2E). Taken together, the data indicate that the increase in the percent of MKI67-positive cells observed in the CL after injection of PGF can be attributed to the CYP11A1-positive, steroidogenic luteal cells and not to the VWF-positive endothelial cells.
Immunohistochemical expression of MKI67 by cells also expressing CYP11A1, a marker of steroidogenesis, and VWF, a marker of endothelial cells. The percent of cells expressing CYP11A1 (A) and the percent of CYP11A1-positive or -negative cells expressing MKI67 (B) were compared at 0, 24, and 48 h after PGF. Representative fields of luteal tissue showing staining for CYP11A1 (red) and MKI67 (green) are shown (C). Nuclei are blue. The percent of cells expressing VWF (D) and the percent of VWF-positive or -negative cells expressing MKI67 (E) were compared at 0, 24, and 48 h after PGF. Representative staining for VWF (red) and MKI67 (green) are shown (F). Staining for MKI67, which is a nuclear antigen, is superimposed over nuclear counterstaining (i.e. not blended with nuclear counterstaining) in order to improve visibility. Data represent the mean±s.e.m. of cell counts from four fields/CL from CL of three cows at each time. Within panels, bars without common superscripts are significantly different (P<0.05). Scale bar=20 μm.
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0324
To determine changes in leukocytes after PGF, sections of CL were stained for the pan leukocyte marker, PTPRC (also known as CD45 or leukocyte common antigen, expressed on all leukocytes). The percent of PTPRC-positive leukocytes in CL sections did not change from 0 to 48 h after PGF (14.8, 17.9, and 14.4% at 0, 24, and 48 h after PGF respectively, P>0.05). Tabulation of the percentages of total luteal cells staining positively for CYP11A1, VWF, and PTPRC in the individual staining procedures showed that the majority of luteal cell types were accounted for in the analyses; the combination of steroidogenic luteal cells, endothelial cells, and leukocytes represented between 96 and 104% of total luteal cells, as depicted in Fig. 3.
The sum of the percent of cells that stained positively for PTPRC (leukocytes), VWF (endothelial cells), and CYP11A1 (steroid-producing cells) at 0, 24, and 48 h after PGF. Immunohistochemistry was performed on separate sections from the same three cows at each time. The data represent the mean±s.e.m. of cell counts of four sections for each antigen from three cows at each time.
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0324
Flow cytometry of cell cycle progression in luteal cells
Flow cytometry was performed to determine the cell cycle and health status of steroidogenic and nonsteroidogenic luteal cells. Dispersed luteal cells were co-stained with Sytox Green, a DNA marker used to assess DNA content, and for Nile Red, a lipid marker used to differentiate relatively lipid-rich steroidogenic luteal cells from nonsteroidogenic luteal cells. Figure 4A shows a representative scatter plot in which Nile Red-positive and -negative cells are identified. Figure 4B shows a representative histogram depicting the number of cells in each phase of the cell cycle (G0/G1, S, and G2/M) based on their DNA content. In addition, apoptotic cells, which have a sub-diploid content of DNA are found in a region known as the A0 peak (Fig. 4B; Darzynkiewicz et al. 1992, Riccardi & Nicoletti 2006). The percent of healthy cells that were Nile Red-positive steroidogenic cells decreased between 24 and 48 h after PGF (Fig. 4C), consistent with the expected loss during luteolysis. The percent of steroidogenic luteal cells in the A0 region increased progressively between 0 and 48 h after PGF (Fig. 4D). By contrast, the percent of nonsteroidogenic luteal cells in the A0 region did not change after PGF (Fig. 4D). Before treatment with PGF, Nile Red-positive steroidogenic luteal cells were predominantly quiescent, with most cells residing in G0/G1 phases of the cell cycle (92%) and few cells in S (7%) or G2/M phases (3%) (Fig. 4E). At 24 h after PGF, the percent of cells in G0/G1 decreased and the percent of cells in G2/M phase increased. At 48 h, the distribution of cells in the cell cycle was similar to that at 24 h. Analysis of the Nile Red-negative nonsteroidogenic cell fraction showed that the distribution in various stages of the cell cycle was not altered after PGF (Fig. 4F). Taken together, the data show that between 0 and 48 h after PGF, cell death occurs in steroidogenic luteal cells concomitantly with reentry of some of these cells into the cell cycle, whereas these changes do not occur in nonsteroidogenic cells. Analysis of the cell cycle by flow cytometry provides information about the population of luteal cells at single time points. Effects of apparently small changes in cell cycle progression and apoptosis observed at single time points would be magnified over time.
The cell cycle distribution of dispersed luteal cells obtained after PGF. Cells were stained with Nile Red, which stains steroid-producing cells, and Sytox Green, which quantitatively binds to DNA. (A) A plot of Nile Red fluorescence vs cellular DNA content (Sytox Green fluorescence) in cells from a CL obtained 24 h after PGF. Cells in the shaded area are considered negative for Nile Red staining; cells above this range are considered positive for Nile Red (i.e. steroid-producing cells). (B) A histogram of cellular DNA content obtained from Nile Red-positive steroid-producing CL cells collected 24 h after PGF. These plots are used to calculate the percent of cells in A0 (apoptotic cells) and in each stage of the cell cycle. The approximate areas of each stage are shown at the top. Based on flow cytometry, the percent of healthy, non-apoptotic luteal cells that were steroidogenic (C) and the percent of steroidogenic and nonsteroidogenic cells that were in the apoptotic A0 region (D) were compared at 0, 24, and 48 h after PGF. The cell cycle distribution of steroidogenic and nonsteroidogenic cells is also shown (E and F). Cell cycle distributions are the percent of all healthy cells; apoptotic cells are not included. All data represent the mean±s.e.m. of luteal cells from three cows at each time. In C and D, bars without common superscripts are significantly different (P<0.05). In E and F, within each stage of the cell cycle, bars without common superscripts are significantly different (P<0.05).
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0324
Manipulation of the cell cycle in cultured luteal cells alters susceptibility to apoptosis
Cultured luteal cells provided a model to test the effects of agents that modulate the cell cycle on susceptibility to apoptosis. Initially, flow cytometry was used to analyze total dispersed luteal cells immediately after isolation on days 12–13 of the cycle and on the same cells after 72 h of culture. Progression through the cell cycle was higher in cultured cells than in freshly isolated cells; the percent of cells in G0/G1 phases were reduced and the percent of cells in S phase was increased (Fig. 5A). Based on Nile Red staining, 42.9±11.9% of cultured cells were steroidogenic cells. Distribution in the cell cycle was assessed for sub-populations of Nile Red-positive and -negative cells (Fig. 5B). Nile Red-positive cells were more highly proliferative than Nile Red-negative cells based on a lower percent of cells in G0/G1 and a higher percent of cells in S and G2/M phases (Fig. 5B). If we focus on S+G2/M cells, which are clearly progressing through the cell cycle, 48% of steroidogenic cells are in S+G2/M while only 13% of nonsteroidogenic cells are in S+G2/M. As 43% of cultured cells are Nile Red-positive steroidogenic cells, it follows that 21% (48% of 43%) of all cells are steroidogenic cells in S+G2/M while 7% (13 of 57%) of all cells are nonsteroidogenic cells in S+G2/M. This suggests that 74% (21% divided by 21+7%) of cells in the cell cycle are steroidogenic cells. These results show that cells that were induced to enter the cell cycle upon culture were predominantly steroidogenic cells.
(A) Cell cycle distribution in freshly isolated luteal cells and in the same cells cultured for 72 h. (B) Cell cycle distribution of cultured luteal cells shown in panel A was recalculated after subdivision into steroidogenic (Nile Red-positive) and nonsteroidogenic (Nile Red-negative) luteal cells. Data represent the mean±s.e.m. of three luteal cell preparations obtained from separate cows. Within each stage of the cell cycle, bars without common superscripts are significantly different (P<0.05).
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0324
In subsequent experiments to examine effects of drugs on susceptibility to FASL-induced apoptosis, Nile Red staining was not analyzed because, as shown above, most cycling cells were steroidogenic and the number of cells available for culture was too low to allow repeatable, multiple-parameter flow cytometry. Cultures were treated with mimosine, which blocks cells in G1 phase, and roscovitine, a CDK inhibitor that blocks cells at the transitions from G1 to S phase and G2 to M phases and cell cycle distribution was examined at 72 h of culture. In cultures treated with either mimosine or roscovitine in the absence of FASL, the percent of cells in the A0 region at 72 h of culture did not differ from controls (Fig. 6A). Mimosine treatment increased the percent of cells in G0/G1 and decreased the percent cells in S relative to control cultures; there was a trend for a decrease in cells in G2/M phases (Fig. 6A). In parallel cultures to test susceptibility of cells to apoptosis, mimosine decreased the cumulative number of cells killed in response to challenge with FASL between 48 and 72 h of culture by 67% compared with that in control cultures (Fig. 6A). Roscovitine increased the percent of cells in G0/G1 and decreased the percent of cells in S phase at 72 h of culture while the percent of cells in G2/M was not changed. In parallel cultures, roscovitine increased the cumulative number of cells killed in response to challenge with FASL between 48 and 72 h of culture by 49% compared with that in control cultures (Fig. 6A). The data indicate that blocking the cell cycle in G1 phase with mimosine protected cells from FASL-induced cell death while blocking at later stages with roscovitine increased susceptibility to cell death.
Cell cycle distribution (left) and killing by FASL (right) in cells cultured with mimosine, roscovitine, or no treatment (control). Data were analyzed in luteal cells obtained 0 h (A) or 24 h (B) after PGF. Mimosine or roscovitine were applied at 48 h of culture, FASL was applied at 52 h, and cells were analyzed at 72 h. Cell cycle distribution and A0 were determined in cells not treated with FASL. Each bar represents the mean±s.e.m. of three cultured luteal cell preparations obtained from separate cows. Within each stage of the cell cycle, and for killing by FASL, bars without common superscripts are significantly different (P<0.05).
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0324
Cell cycle analysis of freshly isolated cells showed that there was an increase in steroidogenic cells progressing through the cell cycle in CL isolated at 24 h after in vivo injection of PGF compared with CL not exposed to PGF (Fig. 4E and F). In order to test whether this difference extended to cells placed in culture and whether it would influence susceptibility to FASL-induced cell death, cultures prepared from CL isolated 0 and 24 h after PGF were studied. The cell cycle distribution at 72 h of culture and effects of inhibitors on the cell cycle did not differ in cultures prepared from CL isolated at 24 h after PGF compared to cultures prepared from CL not exposed to PGF (Fig. 6B compared to A). In addition, the percent of cultured cells killed by challenge with FASL between 48 and 72 h of culture were similar in cells isolated before and after PGF treatment in vivo both in control media and in media containing mimosine and roscovitine (Fig. 6B compared to A). These results indicate that culturing luteal cells induces them to enter the cell cycle. Previous exposure to PGF in vivo does not alter the cell cycle distribution of cultured cells and does not affect the susceptibility of cultured luteal cells to FASL-induced death. These findings are consistent with an important role of the cell cycle in determining susceptibility to apoptosis.
As an additional approach to test the effect of the cell cycle on susceptibility to apoptosis, dispersed luteal cells isolated on days 12–13 of the cycle were cultured and infected with recombinant adenoviruses (Ad) expressing cyclin-dependent kinase inhibitors CDKN1A or CDKN1B, or with a null adenovirus that does not express a recombinant protein (AdNull). In cultures infected with adenoviruses in the absence of FASL, the percent of cells in A0 increased in response to AdCDKN1A and were unchanged in response to AdCDKN1B or AdNull (Fig. 7). Infection with AdCDKN1A or AdCDKN1B increased the percent of cells in G0/G1 and decreased the percent of cells in S and G2/M phases relative to control cultures (Fig. 7). Infection with AdNull also increased the percent of cells in G0/G1 and decreased cells in S relative to control cultures, although the changes were not as pronounced as in response to AdCDKN1A or AdCDKN1B and there was no effect on the percent of cells in G2/M. While AdCDKN1A and AdNull increased the percent cell death in response to FASL over 24 h, AdCDKN1B decreased FASL-induced cell death. These data indicate that while both AdCDKN1A and AdCDKN1B decreased progression through the cell cycle, only AdCDKN1B protected against FASL-induced cell death.
Cell cycle distribution (left) and killing by FASL (right) in cells cultured with AdCDKN1A, AdCDKN1B, AdNull or no adenovirus (control). Adenovirus was applied at 24 h of culture, FASL was applied at 52 h, and cells were analyzed at 72 h. Cell cycle distribution and A0 were determined in cells not treated with FASL. Each bar represents the mean±s.e.m. of three cultured luteal cell preparations obtained from separate cows. Within each stage of the cell cycle, and for killing by FASL, bars without common superscripts are significantly different (P<0.05).
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0324
Effect of progesterone on the cell cycle and susceptibility to apoptosis in vitro
The effect of progesterone and the PGR antagonist RU486 on progression through the cell cycle and susceptibility to FASL-induced cell death was tested in cultured luteal cells from days 12–13 CL. Endogenous progesterone in media harvested from control cultures was considerable after 24 and 48 h, but at 72 h decreased to only 18% of the concentration measured at 48 h (Fig. 8A). This drop in endogenous progesterone may be due to the fact that culture media contained serum for the first 48 h but not from 48 to 72 h (described in Materials and Methods). Addition of exogenous progesterone significantly increased the concentration of progesterone in media (Fig. 8A) and blunted the decrease at 72 h of culture. Cell cycle analysis at 72 h of culture showed that progesterone increased the percent of cells in G0/G1 and decreased cells in S and G2/M phases relative to control cultures (Fig. 8B). Treatment with RU486 alone did not alter the distribution of cells in the cell cycle but co-treatment with progesterone and RU486 blocked the effect of progesterone. Progesterone decreased the percent cell death in response to challenge with FASL over 24 h while treatment with RU486 blocked this effect (Fig. 8B). While effects of progesterone on the cell cycle at the 72 h time point appear small, effects would be cumulative over time in culture, and could contribute to the dramatic protection against FASL-induced apoptosis over 24 h of culture. The data suggest that a PGR-mediated effect of progesterone to decrease progression through the cell cycle is associated with its protective effect against FASL-induced cell death.
Effect of progesterone on the cell cycle and susceptibility to FASL-induced killing. Panel A shows the effect of treatments with progesterone (P4), the progesterone receptor antagonist RU486, progesterone plus RU486, or no treatment (control) on the concentration of progesterone measured in culture media. Panel B shows cell cycle distribution and A0 in the absence of FASL (left) and killing in response to challenge with FASL (right) in the presence of various treatments. Treatments were applied at 0, 24, and 48 h of culture, FASL was applied at 52 h, and cells were analyzed at 72 h. Each bar represents the mean±s.e.m. of three cultured luteal cell preparations obtained from separate cows. Within each stage of the cell cycle, for killing by FASL, and for progesterone over 24–72 h of culture, bars without common superscripts are significantly different (P<0.05).
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0324
Effect of nitric oxide altering reagents on the cell cycle and susceptibility to apoptosis in vitro
Increased production of nitric oxide by treatment with reagents such as spermine–nitric oxide complex (NONO), a nitric oxide donor, has been associated with luteolysis, while treatment with inhibitors of nitric oxide synthesis such as Nω-nitro-l-arginine methyl ester hydrochloride (NAME), have been shown to extend the life of the CL (reviewed in Discussion). It was of interest to determine whether the effects of these reagents on luteal cell survival were associated with changes in the cell cycle. In cells from days 12–13 CL, NONO and NAME did not alter progression through the cell cycle (Fig. 9). While NAME reduced FASL-induced cell death over 24 h by 94%, NONO had no effect (Fig. 9). The data indicate a lack of association between the protective effects of NAME against FASL-induced cell death and progression through the cell cycle.
Cell cycle distribution (left) and killing by FASL (right) in cells cultured with NAME (a nitric oxide antagonist), NONO (a nitric oxide agonist), or no treatment (control). NAME was applied at 0, 24, and 48 h of culture, NONO was applied at 48 h, FASL was applied at 52 h, and cells were analyzed at 72 h. Cell cycle distribution and A0 were determined in cells not treated with FASL. Each bar represents the mean±s.e.m. of three cultured luteal cell preparations obtained from separate cows. Within each stage of the cell cycle, and for killing by FASL, bars without common superscripts are significantly different (P<0.05).
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0324
Discussion
A major finding of the current study is that quiescent, differentiated steroidogenic cells in the fully developed CL reenter the cell cycle during luteal regression. Reentry into the cell cycle is associated with deletion of these cells by apoptosis. Placing luteal cells in culture promoted reentry into the cell cycle, and blocking cultured cells in G0/G1 phase by treatment with cell cycle inhibitors or progesterone promoted resistance to apoptosis.
The number of Nile Red-positive steroidogenic cells was not altered between 0 and 24 h after PGF but had declined by 48 h (Fig. 4C). This is consistent with a previous study in which plasma progesterone decreased between 4 and 8 h after injection of cows with a luteolytic dose of PGF while weight of the CL was unchanged through 24 h and decreased between 24 and 48 h, and apoptotic fragmentation of DNA was not readily detectable until 24 h after PGF (Juengel et al. 1993). Histological analysis and analysis of the cell cycle by flow cytometry indicated that few steroidogenic cells were proliferating in the mid-cycle CL (Figs 2B and 4E). After injection of PGF, the number of steroidogenic cells progressing through the cell cycle and undergoing apoptosis increased (Figs 2B and 4D, E). In addition, after PGF, a fraction of cells co-stained positively for the proliferation marker MKI67 and the apoptosis marker cPARP1 (21% of MKI67-positive cells were cPARP1 positive; Fig. 1C), suggesting that cells enter the cell cycle before initiation of apoptosis. The fact that a small fraction of MKI67-negative cells were positive for apoptosis (9.9% of MKI67-negative cells were cPARP1 positive, Fig. 1C) may indicate that some cells undergo apoptosis without reentering the cell cycle and suggests that cell cycle entry may not be obligatory for apoptosis. Alternatively, in cPARP1-expressing cells in later stages of apoptosis, proteins may be lost as apoptotic cells condense, and expression of proteins like MKI67 may not occur.
Several previous studies reported expression of cell proliferation markers during luteal regression but the biological significance of the findings was not explained. In rats, luteal cells were positive for the cell proliferation marker, PCNA, during structural regression of the CL (Kiya et al. 1998, 1999). In the mare, MKI67-positive steroidogenic luteal cells were observed late in the process of natural luteal regression and at 36 h after injection of a PGF analog (Al-zi'abi et al. 2003). In a subsequent study, positive staining for a marker of mitosis, phosphorylated histone-3, was observed at 36 h after injection of mares with a PGF analog, a time point when many luteal cells were undergoing cell death (Aguilar et al. 2006). A number of studies have shown increased expression of early response genes involved in cell proliferation during luteolysis; the relationship between these changes and regression is not known. Expression of early response genes increased in response to PGF in pig CL that had acquired luteolytic capacity but not in CL of the early cycle that were refractory to PGF (Diaz et al. 2013). PGF treatment also increased expression of early response genes in the bovine CL (Betrand & Stormshak 1996, Tsai et al. 2001). In a recent study, cows were given intrauterine infusions of PGF to produce multiple low-dose pulses of PGF that mimic the pattern in natural luteal regression and multiple biopsies were collected from individual CL to examine temporal changes in gene expression; levels of mRNA for immediate early genes increased after each pulse of PGF, suggesting a role in luteolysis (Atli et al. 2012).
Our finding that steroidogenic luteal cells enter the cell cycle when placed in culture (Fig. 5) provided a model to study the connection between entry into the cell cycle and susceptibility to apoptosis. Interestingly, there was no difference in cell cycle distribution or the response to treatment with FASL in cultured luteal cells derived from mid-cycle CL compared with luteal cells isolated 24 h after injection of cows with PGF. In addition, the response to altering the cell cycle with various drugs was similar (Fig. 6A and B). It seems likely that cells that are viable at the time of isolation have the potential to survive in culture because factors that might cause their demise in vivo are absent in vitro.
When luteal cells were cultured with a cell cycle inhibitor that induced them to remain in G1 phase, they became resistant to apoptosis, supporting the concept that cells are relatively resistant to apoptosis when in G0 or G1 phase of the cell cycle. Mimosine, an inhibitor known to block cells in G1 phase, before the G1/S phase transition (Lalande 1990, Krude 1999), decreased progression of luteal cells through the cell cycle and protected against FASL-induced death relative to untreated control cultures (Fig. 6A and B). By contrast, roscovitine, a CDK2 inhibitor that blocks transition from G1 to S phase and from G2 to M, increased FASL-induced apoptosis (Fig. 6A and B). It is possible that by blocking the S to G2/M transition, roscovitine maintains a higher percentage of cells in a susceptible state.
In mice, luteinization is associated with decreased expression of positive regulators of the cell cycle, CDK1 and cyclin D1, and formation of protein trimers consisting of CDKN1B (p27KIP1), cyclin D3, and CDK4, changes that are thought to be involved in maintenance of the post-mitotic state and luteal phenotype (Hampl et al. 2000). Mice null for CDKN1B are infertile, withdrawal of granulosa cells from the cell cycle after the LH surge is delayed, and proliferative cells are present in the early CL (Kiyokawa et al. 1996). Mice null for CDKN1A (p21WAF1/CIP1) are fertile with no obvious ovarian phenotype (Brugarolas et al. 1995, Deng et al. 1995). Mice double null for CDKN1A and CDKN1B have pronounced extension of proliferation of cells in the developing CL, more than observed in CDKN1B single null mice, and an abnormally high level of luteal cell apoptosis (Jirawatnotai et al. 2003). These findings suggest that in luteal cells, CDKN1B plays the predominant role in maintaining quiescence, while the role of CDKN1A may be supportive but less critical. In addition, they are consistent with the concept introduced in the current study that the quiescent state of steroidogenic cells in the functional CL promotes prolonged luteal cell survival while proliferative activity supports apoptosis.
Infection of luteal cells with AdCDKN1B (p27KIP1) decreased progression through the cell cycle and protected cells from FASL-induced cell death, findings consistent with a protective effect of cell cycle exit on luteal cell survival. By contrast, infection with AdCDKN1A (p21WAF1/CIP1) increased FASL-induced killing (Fig. 7). While treatment with AdCDKN1A reduced progression through the cell cycle, it also increased the percent cells in the A0 apoptotic peak and this cytotoxicity may have contributed to increased cell death in response to FASL. Control cells treated with AdNull had higher cell killing in response to FASL than untreated controls, suggesting a small effect of adenovirus infection. The reasons for the differences in effects of the two cell cycle inhibitors in vitro are not known, but the results are interesting in light of the more prominent effect of CDKN1B compared with CDKN1A in maintaining quiescence of luteal cells in vivo, as revealed by the studies in genetically modified mice described earlier (Hampl et al. 2000, Jirawatnotai et al. 2003).
The in vitro studies reported here showed that progesterone inhibited progression of luteal cells through the cell cycle and protected cells against FASL-induced cell death, effects that were prevented by co-treatment with RU486 and that are therefore likely to be mediated through PGR (Fig. 8). Previous studies support a role for progesterone in maintaining luteal function: treatment of bovine luteal cells with a PGR antagonist or an inhibitor of progesterone production increased the rate of spontaneous apoptosis in vitro (Rueda et al. 2000, Okuda et al. 2004, Liszewska et al. 2005). Treatment of rhesus monkeys with an inhibitor of progesterone production during the luteal phase blocked the effect of hCG to maintain luteal weight (Duffy & Stouffer 1997). In addition, we previously showed that bovine granulosa cells became resistant to apoptosis after exposure to the LH surge and that this effect was mediated by an effect of PGR to maintain cell quiescence (Quirk et al. 2004). Progesterone also had an inhibitory effect on proliferation of human granulosa cells (Chaffkin et al. 1992). In the context of events in vivo, the decline in progesterone during functional regression of the CL may promote reentry of cells into the cell cycle and this may be one of the mechanisms whereby susceptibility to apoptosis is increased.
Luteal regression involves the activation of cytotoxic pathways. The production of reactive oxygen species increases and the expression of genes involved in protecting against oxidative stress decreases during luteolysis (Rueda et al. 1995). In cultured luteal cells, the production of reactive oxygen species increased oxidative damage, expression of Trp53 (p53), and apoptosis (Nakamura & Sakamoto 2001). Along these lines, reentry into the cell cycle and apoptosis in various neurodegenerative diseases are associated with increased oxidative stress (Klein & Ackerman 2003). Luteal blood flow and plasma progesterone declined at luteolysis in cows (Acosta et al. 2002, Nishimura et al. 2008) and hypoxia reduced progesterone synthesis (Nishimura et al. 2006) and caused apoptosis of bovine luteal cells (Nishimura et al. 2008). Pro-apoptotic proteins such as the cytokines tumor necrosis factor α and FASL increase during luteolysis and are believed to contribute to structural regression (Shaw & Britt 1995, Friedman et al. 2000, Quirk et al. 2000b, Petroff et al. 2001, Taniguchi et al. 2002, Carambula et al. 2003, Okuda & Sakumoto 2003, Skarzynski et al. 2003, Galvao et al. 2010, Atli et al. 2012). The results of the current study suggest that luteal cells reenter the cell cycle and become susceptible to apoptosis during the same time period when cytotoxic pathways are activated in vivo.
Production of nitric oxide may be involved in luteolysis because it is produced by luteal cells, increases at the time of luteolysis, and decreases progesterone production by cells in vitro (Jaroszewski et al. 2003a, Skarzynski et al. 2003, 2007, Neuvians et al. 2004). In addition, treatment of cows with a nitric oxide synthase inhibitor delayed luteal regression (Jaroszewski & Hansel 2000, Skarzynski et al. 2003). Overall, the findings of the current study are consistent with a negative effect of nitric oxide on luteal cell survival because preventing production of nitric oxide with the inhibitor NAME protected cells from FASL-induced cell death. However, NAME had no effect on progression through the cell cycle, indicating that its protective effect was not dependent on maintaining luteal cells in a quiescent state (Fig. 9). It is believed that the decrease in progesterone production by the CL in response to PGF in vivo is caused by effects on multiple cell types mediated through autocrine and paracrine factors including nitric oxide (Skarzynski et al. 2000, 2007, Jaroszewski et al. 2003b, Korzekwa et al. 2004). While treatment of cultured bovine luteal cells with a nitric oxide donor was previously shown to increase the percent of cells with fragmented DNA (Korzekwa et al. 2006), in the current study, treatment with NONO had no effect on FASL-induced cell death.
Recognizing that reentry into the cell cycle promotes the susceptibility of luteal cells to apoptosis advances our understanding of the multiple factors involved in luteal regression. Our data suggest that reentry of luteal cells into the cell cycle is an early step in the process of luteolysis. It remains to be determined whether the resistance of the CL to PGF-induced luteolysis during the early estrous cycle is related to inability of cells to reenter the cell cycle. While reentry into the cell cycle is known to be associated with cell death in pathological situations such as neural degeneration, the current study establishes the CL as a physiological model for the influence of the cell cycle on deletion of differentiated cells within a regressing tissue.
Materials and Methods
Animals
All procedures were approved by the Cornell University Institutional Animal Care and use Committee and conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Non-lactating luteal phase Holstein cows were injected with 30 mg PGF (Lutalyse, Pfizer Animal Health, New York, NY, USA) to induce estrus and initiate a timed estrous cycle. The presence of a CL was confirmed by transvaginal ultrasonography at least three times between days 7 and 12, including the day before ovariectomy. On day 12 or 13 after standing heat, cows were injected with 30 mg PGF to induce luteolysis. Luteolysis was confirmed by RIA of progesterone in plasma samples obtained 0, 12, 24, and 48 h after injection. The ovary bearing the CL was removed by colpotomy (Drost et al. 1992) at 0, 24, or 48 h after PGF. Ovaries were placed in PBS and transported to the laboratory within 15 min.
Tissue preparation
The CL was dissected from the ovary, cut into pieces, and several pieces placed in HistoPrep embedding media (Fisher Scientific, Pittsburgh, PA, USA) and frozen in liquid nitrogen for subsequent immunohistochemistry. Remaining luteal tissue was used to isolate dispersed cells for culture and for use in flow cytometry. Tissue digestion was based on the method of Pate & Condon (1982), with modifications. Two to 4 g luteal tissue was minced with a razor blade and digested in a 50 ml tube with 200 U collagenase (Type I, Gibco, Life Technologies) per gram of luteal tissue in Ham's F-12 media containing 0.5% BSA and 24 mM HEPES (all Gibco, Life Technologies). Tubes were rocked at 37 °C and triturated every 15 m. After 45 m, tissue was passed through cotton gauze and dispersed material held aside. Remaining tissue was further digested in 4 mg/ml collagenase/dispase (Roche Diagnostics) plus 10 μg/ml DNase I (Sigma–Aldrich) in Ham's F-12–BSA–HEPES for 45 m with trituration every 15 m. Dispersed cells were combined, passed through gauze, and centrifuged at 400 g. The pellet was resuspended in 1 ml Ham's F12 media and cell number determined. Cells for flow cytometry were fixed in ice-cold 80% ethanol. Cells for culture are described below.
Immunohistochemistry
Eight micrometer-thick cryostat sections were fixed in 2% paraformaldehyde, rinsed in PBS, and blocked in PBS containing 2% normal goat serum. Sections were incubated with primary antibodies in PBS-1% BSA. Sections were rinsed in PBS, and Alexa 488-conjugated goat anti-mouse IgG or Alexa 555-conjugated goat anti-rabbit IgG (Molecular Probes, Life Technologies) were used to detect the antibodies. Cell nuclei were counterstained with Hoechst 33342 (Molecular Probes, Life Technologies). Antibodies used were mouse anti-human MKI67 (clone MM1, Vector Laboratories, Burlingame, CA, USA), rabbit anti-human cPARP1 (specific for PARP1 cleaved at Asp214, which occurs during apoptosis; Ab #9541, Cell Signaling Technology, Beverly, MA, USA), rabbit anti-rat CYP11A1 (AB1244, Chemicon International, Temecula, CA, USA), rabbit anti-human VWF (expressed by endothelial cells, some platelets, and megalokaryocytes; product #F3520, Sigma–Aldrich), and mouse anti-bovine PTPRC (clone CC1, Biosource, Invitrogen, Grand Island, NY, USA). Antibodies to cPARP1, CYP11A1, and VWF were each used in co-staining procedures with antibody to MKI67.
Stained sections were observed at 40× magnification under epifluorescent illumination, and coincident digital images of relevant staining, covering an area of 355×279 μm, were obtained using a Spot II camera (Diagnostic Instruments, Sterling Heights, MI, USA). Color and brightness were adjusted using Adobe Photoshop; identical settings were used and saved for all images of each type of staining. For each antigen, the percent of cells positively stained were calculated as follows: an image of staining for the specific antigen was overlaid with an image of Hoechst nuclear staining. A slash was placed over the nucleus of each positively stained cell. When analyzing co-staining for a second antigen, slashes oriented in the opposite direction were used to mark nuclei of positively stained cells, and marked nuclear images were overlaid to determine co-staining. Marked nuclei and total nuclei were counted and percent positive calculated. The individual counting stained cells was unaware of sample identification. Each type of staining was performed on three animals each at 0, 24, and 48 h after PGF. Four fields were examined from each animal for each type of staining; fields contained an average of 366 cells each (range 234–540 cells/field). In images shown in figures, staining for nuclear antigens MKI67 and cPARP1 are superimposed over nuclear counterstaining (i.e. not blended with nuclear counterstaining) in order to improve visibility of MKI67-stained and cPARP1-stained nuclei.
Cell culture
Dispersed luteal cells isolated from cows at 0 or 24 h after PGF were cultured and analyzed for susceptibility to apoptosis and for cell cycle distribution. Experimental treatments were added to the appropriate media at various times and are described below; the media used in all experiments were the same. All media reagents were from Gibco, Life Technologies. Dispersed cells were plated in Ham's F-12 medium (supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml fungizone, 1 mM pyruvate, and 2 mM glutamine) containing 10% FBS at a density of 5×104 cells/well in 96-well culture plates or 1×106 cells/well in six-well culture plates. After 24 h, media were changed to DMEM/F-12 media supplemented as above and containing 10% FBS. At 48 h, media were changed to serum-free DMEM/F-12 supplemented as above and also containing 100 ng/ml insulin, 5 μg/ml transferrin, 20 nM sodium selenite, and 0.1% BSA. The concentration of insulin in this media is significantly lower than in media prepared with commercial ‘ITS’ preparations. While the presence of serum in media can block the effects of apoptosis-inducing agents, the use of media that contains a low level of ITS allows us to test the susceptibility of cells to apoptosis (Quirk et al. 2000a, Hu et al. 2004). To determine susceptibility to apoptosis, cells cultured in 96-well plates were treated at 52 h with 0 or 100 ng/ml soluble recombinant human FASL (Enzo Life Sciences, Farmingdale, NY, USA). At 72 h, the number of viable cells attached to each well was determined: cells were removed from plates by treatment with trypsin, collected and stained with trypan blue, and live cells were counted in a hemacytometer. The percent of luteal cells undergoing FASL-induced cell death were calculated by comparing the number of live cells present in FASL-treated cultures vs the number of cells in control cultures receiving no FASL but otherwise treated identically. Experiments were repeated three times using cultures from different cows, and treatments within each replicate were performed in quadruplicate wells. To determine the effects of treatments on the cell cycle, cells that were cultured in six-well plates were harvested at 72 h, fixed in ice-cold 80% ethanol, and subsequently analyzed for DNA content by flow cytometry. All cell cycle experiments were repeated three times using cultures from different cows.
In the first culture experiment, cells were treated at 48 h with cell cycle inhibitors: 1 mM mimosine (Calbiochem), 20 μM roscovitine (Calbiochem, EMD Millipore, Billerica, MA, USA), or no treatment (control). Doses were based on our previous studies with bovine granulosa cells (Hu et al. 2004, Quirk et al. 2004, 2006). Treated cells were analyzed for effects on the cell cycle and susceptibility to apoptosis as described earlier. In the second experiment, cells were treated at 24 h with 10 pfu adenovirus containing genes for the cyclin-dependent kinase inhibitors CDKN1A (AdCDKN1A) (Katayose et al. 1995, Quaroni et al. 2000), or CDKN1B (AdCDKN1B) (Craig et al. 1997, Quaroni et al. 2000), obtained from Dr A Quaroni, Cornell University. Control cultures were not infected with adenovirus or infected with 10 pfu AdNull. In the third experiment, cells were treated with 1 μg/ml progesterone (Sigma–Aldrich), 0.44 μg/ml RU486 (mifepristone, Sigma–Aldrich), or no treatment. Doses were based on our previous studies with bovine granulosa cells (Quirk et al. 2004). Treatments were applied at 0, 24, and 48 h. Media from 96-well plates in this experiment were saved and assayed for progesterone using a Progesterone EIA from Cayman Chemical (Ann Arbor, MI, USA). Within each replicate, media from individual wells for each treatment were pooled and assayed in duplicate. In the final culture experiment, cells were treated at 0, 24, and 48 h with 10 μM NAME (Sigma–Aldrich), or at 48 h with 100 μM NONO (Spermine NONOate, Sigma–Aldrich). Doses were chosen based on previous studies with bovine luteal cells (Jaroszewski et al. 2003a, Korzekwa et al. 2004).
Cell cycle analysis by flow cytometry
Stage of the cell cycle was determined by measurement of the DNA content of cells by staining with Sytox Green (Molecular Probes, Life Technologies) and analysis by flow cytometry. In some experiments, cells were co-stained with Nile Red (Sigma–Aldrich), which binds to neutral lipids and distinguishes steroidogenic luteal cells that have high lipid content from nonsteroidogenic cells that have lower lipid content. Cells were stained with 1 μM Sytox Green plus 5 μg/ml Nile Red (where used) in 0.01 M PBS containing 0.01% Triton X-100 and 30 μg/ml deoxyribonuclease-free ribonuclease A (Sigma–Aldrich). Cells (10 000 per sample) were analyzed on a FACScan flow cytometer (Becton Dickinson and Co., Franklin Lakes, NJ, USA). Data were gated for single cells, and DNA content, based on Sytox Green fluorescence, was used to assign cells to A0, G0/G1, S, or G2/M phases based on the method of Ormerod (1994) using WinMDI software (The Scripps Research Institute, La Jolla, CA, USA). In cells also stained with Nile Red, cells (20 000 per sample) were gated as Nile Red-positive or -negative before DNA analysis. In analysis of the flow cytometry data, the area of Nile Red-negative cells was selected to contain 95% of cells in a sample not stained with Nile Red; cells with fluorescence higher than this were considered Nile Red-positive. The percent of cells in A0 were calculated as percent of all cells; percent of cells in G0/G1, S, or G2/M were calculated as the percent of non-A0 cells.
Statistical analysis
The results of culture experiments were analyzed by randomized complete block ANOVA, where individual culture preparations are blocks and received all treatments within a given experiment. Progesterone concentrations were log transformed before analysis. Cell cycle stages in freshly isolated vs cultured luteal cells were analyzed by paired t-test. All other data were analyzed by simple one-way ANOVA.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This project was supported by National Research Initiative Competitive Grant 2004-35203-14810 from the USDA Cooperative State Research, Education, and Extension Service.
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