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Involvement of cell proliferation in the process of follicular atresia in the guinea pig
Tissue and Cell 42 (2010) 234–241
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Involvement of cell proliferation in the process of follicular atresia in the guinea pig Wei Wang a , Honglin Liu a , Wei Ding a,b , Yan Gong a , Jingwei Chen a , Reinhold J. Hutz c , Dagan Mao a , Fangxiong Shi a,∗ a b c
Laboratory of Animal Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China Department of Animal Husbandry and Veterinary Medicine, Jiangsu Polytechnic College of Agriculture and Forestry, Jurong 212400, PR China Department of Biological Sciences, University of Wisconsin-Milwaukee, WI 53201, USA
a r t i c l e
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Article history: Received 20 January 2010 Received in revised form 21 April 2010 Accepted 27 April 2010 Available online 3 June 2010 Keywords: Follicular atresia Cell proliferation Guinea pig
a b s t r a c t Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction A considerable amount of research has been performed on follicular atresia during the last decade, and the main work focused on cell death and elimination (Alonso-Pozos et al., 2003; Nezis et al., 2006; Santos et al., 2008). However, little was known about the cell proliferation in the process of follicular atresia. In our current paper, these phenomena were documented in detail in guinea pigs. In view of some morphologic characteristics described for the first time, several special terms were cited based on the similarity of structures and functions. In the process of follicular atresia, the morphologic changes were obvious, and some polymorphic cells are differentiated from the innermost theca layer, which were described as “stellate or slender cells” (Kasuya, 1997), “fibroblastoid cells”, “fibroblast-like cells” and “fibroblastoid-looking cells” (Logothetopoulos et al., 1995). In the present paper, the polymorphic cells were denominated as “fibroblast-like cells” (Wang et al., 2010).
∗ Corresponding author. E-mail addresses: [email protected] (W. Wang), [email protected] (F. Shi). 0040-8166/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2010.04.006
It had been reported that a large numbers of fibroblast-like cells appeared in the antrum of atretic follicles, and documented as a “cellular network” (Kasuya, 1997). However, cell proliferation was not confirmed (Kasuya, 1997; Logothetopoulos et al., 1995). In the present study, the protein proliferating cell nuclear antigen (PCNA) was used as the proliferation marker (Bolton et al., 1994; Paunesku et al., 2001; Wildemann et al., 2003). PCNA – a cofactor of DNA polymerases that encircles DNA – orchestrates several of these functions by recruiting crucial players to the replication fork (Moldovan et al., 2007). Our results showed that cell proliferation did occur in atretic follicles. In addition, the term “cellular network” was not included in the changes within the follicular antrum. When a large number of fibroblast-like cells appeared, the antrum was not empty any more, and had very loose connections. This structure was described as “loose connective tissue” (Reed et al., 2009; Tzouvelekis et al., 2009; Weber, 1999; Wolf et al., 2009). Subsequently, with the proliferation of the differentiated cells, a new tissue formed in the antrum, which was described as “new tissue” (Leite et al., 2002). Although obvious changes occurred in the theca layers, the follicles were destined to degenerate. With the shrinking of follicular volume, atretic follicles lost their identifications (Logothetopoulos et al., 1995). Cellular morphologic changes during follicular atresia were fully investigated in the present study using histology, and we confirmed
W. Wang et al. / Tissue and Cell 42 (2010) 234–241
follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation.
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Twenty-five adult female guinea pigs (Cavia porcellus) of the Hartley strain were used at 5 months of age, with an initial weight of 400–700 g. They were housed 4 animals per cage, under controlled temperature at 23 ± 2 ◦ C, and fed commercial food and tap water ad libitum. Estrous cycles were recorded by daily examination of vaginal smears whenever the vagina was open (Shi et al., 1999). The day of ovulation was estimated as the day when the maximal cornification was seen in the smear (Hutz et al., 1990; Lilley et al., 1997; Norris and Adams, 1979) and was designated as day 0 of the cycle. Days of the estrous cycle followed thereafter consecutively. We used only animals showing at least two consecutive 15–17-day cycles immediately prior to the experiment. The animals were sacrificed on days 1, 4, 8, 12, and 16 (5 animals per day) (Garris and Mitchell, 1979). Then the ovaries were collected immediately for experiments.
of L1 (diameters in centripetal direction) to L2 (diameters in tangent direction) in the nuclei of theca interna cells were measured for evaluating the follicular atresia. It was widely accepted that the follicular growth of guinea pigs was biphasic (Bland, 1980; Fortune, 1994; Hutz et al., 1990; Shi et al., 1999). In the current study, follicles on days 16, 1 and 4 were investigated, which constituted the second wave of follicular growth and atresia. Only those follicles with the largest cross-sectional area were measured, and we ensured that each follicle was measured only once. The sample size of each group was at least twenty. Microscopic measurements were made with a Nikon 80i (Nikon, Tokyo, Japan). Follicular diameter was taken as the mean of the largest diameter and the diameter perpendicular to the midpoint (Bland, 1980). Thickness of theca interna cells was measured at three different positions, and the mean was used for statistical analyses. The ratio of L1 to L2 was taken to denote the arrangement changes in the nuclei of theca interna cells, and the migrated fibroblast-like cells which had very thin shapes were excluded. At least three areas at 50 m × 50 m were measured in each follicle, and the mean was used for statistical analyses. Therefore, the ratio more than or less than 1.0, respectively, represented the arrangement of theca interna cells in centripetal or tangent direction.
2.2. Reagents
3. Results
PCNA monoclonal antibodies were purchased from Biogenix Co. (San Ramon, CA, USA, lot: Mu2060899); immunohistochemical kits (SABC methods) were purchased from Boshide Co. (Wuhan, Hubei, China, lot: SA1021); 3,3 -diaminobenzidine tetrachloride (DAB) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were purchased reagent grade.
3.1. Follicular atresia in normal estrous cycles
2. Materials and methods 2.1. Animals and sample collection
2.3. Histological and immunohistological studies Ovaries were fixed in 4% paraformaldehyde at room temperature for 24 h, embedded in paraffin, sectioned serially at 10 m and stained with hematoxylin and eosin (HE). These sections were analyzed for the morphologic changes indicative of atretic follicles. To examine the proliferating cells in the atretic follicles, immunohistochemical staining was performed using monoclonal antibodies to PCNA with the streptavidin–biotin-complex (SABC) method. A few sections were picked randomly from the serial sections. Sections were mounted on slides coated with APES and which were dried for 24 h at 37 ◦ C. The antibodies were diluted 1:500 in PBS containing 1% bovine serum albumin (BSA), and the sections were incubated overnight at 4 ◦ C with primary antibodies. The specific protein immunoreactivity was visualized by 0.05% DAB in 10 mM PBS-buffered saline containing 0.01% H2 O2 for 2–5 min, and counterstained with hematoxylin. A negative control was a primary antibody blank, and relative levels of immunostaining were evaluated and repeated at least four times. The percent area of follicular cells staining positive for PCNA by more than 50% were considered strongly positive, between 10 and 50% were considered positive, and less than 10% were considered to be negative. The images were captured in a Nikon 80i (Nikon, Tokyo, Japan). Three areas at 50 m × 50 m were selected randomly for evaluation within each follicle, and at least six follicles were evaluated for each stage of follicular atresia. 2.4. Evaluation of follicular atresia Follicles were considered as round shape in the sections, and cellular arrangement was assigned in centripetal or tangent direction. The tangent direction was perpendicular to centripetal direction. Follicular diameter, thickness of theca interna cells, and the ratio
On day 1 after ovulation, there were very few healthy antral follicles in the ovaries, and the new corpora lutea were observed. A mass of dead granulosa cells separated from the granulosa layers and scattered in the antrum. Furthermore, in some atretic follicles where granulosa cells were eliminated, fibroblast-like cells appeared in the theca interna cells. On day 4, the most atretic follicles were shrunken, and the follicular antrum was filled with fibroblast-like cells. Some atretic follicles exhibited a very small volume and the fibroblast-like cells disappeared. On days 8 and 12, a group of large antral follicles appeared. Many of these acquired large population of granulosa cells. However, the antral follicles were often in early stages of atresia. On day 16, antral follicles were further developed. Ovaries at this time contained 0–3 unruptured preovulatory follicles, which had diameters at least 700 m. Although many follicles showed large volumes, there was a mass of desquamated granulosa cells and were distributed along the follicular cavity. At this time, it was obvious that the intensity of the follicular atresia was greater than on either day 8 or 12. 3.2. Cell elimination in follicular atresia Based on the morphologic observations (Figs. 1 and 2), there were continuous changes in the process of follicle atresia in guinea pigs. The most remarkable characteristics included the elimination of granulosa cells and the formation of new tissue. Moreover, it seemed that the fibroblast-like cells played a central role in new tissue formation. In healthy follicles, granulosa cells were arranged neatly (Fig. 1A and Fig. 2A). When the atretic process began, connections in the granulosa layers became loose, and the columnar granulosa cells became round (Fig. 2B), with many pyknotic cells and apoptosis bodies were observed (Fig. 2C). With the massive elimination of granulosa cells, granulosa layers disintegrated gradually, and dead granulosa cells were shed into the follicular antrum (Fig. 1B). At the same time, the fibroblast-like cells appeared in the innermost layer of theca cells (Fig. 2D).
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Fig. 1. Morphologic observations on follicular atresia. In healthy follicles, granulosa cells were arranged neatly (A). With the granulosa cells eliminated (B), the fibroblast-like cells appeared and invaded the antrum (C), and formed a type of loose connective tissue (D). Meanwhile, more and more cells congregated outside the zona pellucida (E), and formed a new tissue in the antrum (F). Subsequently, the new tissue degenerated in a manner similar to hyalinization (G), and the loose connective tissue appeared again (H). Finally, the follicle exhibited a very small volume (I). Arrowheads represent dead granulosa cells; arrows represent fibroblast cells. O, oocyte; ZP, zona pellucida; G, granulosa layer; T, theca layer; A, follicular antrum; LCT, loose connective tissue; NT, new tissue. Bar = 50 m.
After the complete elimination of granulosa cells, the innermost layer of theca cells had fibroblast-like shapes (Fig. 1C and Fig. 2E). Later, the fibroblast-like cells appeared in the follicular antrum solely, and the leading edge extended very long protrusions to the antrum (Fig. 2F). Subsequently, a kind of loose connective tissue invaded the whole antrum subsequently. The antrum was then no
longer empty at that period, but filled with many cells and ECM (Fig. 1D). Call–Exner bodies sometimes appeared in the antrum, while the cells close to the antrum were arranged in a centripetal orientation (Fig. 1E). With more and more fibroblast-like cells appeared in the antrum, a mass of cells congregated surrounded the zona pellucida,
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Fig. 2. Morphologic observations on theca interna cells. In healthy follicles, the theca interna cells manifested an orderly nucleus, and were arranged in the same orientation (A). With the process of atresia (B and C), dead granulosa cells were shed into the follicle antrum, and fibroblast-like cells appeared in the innermost layer of theca cells (D). Later, many fibroblast-like cells appeared in the theca layer (E) and the antrum (F). When fibroblast-like cells were few in the theca layer, some intumescent cells appeared in the antrum (G). Subsequently, the follicular volume shrank, and the fibroblast-like cells appeared again (H). Finally, only a few layers of theca interna cells remained, and the fibroblast-like cells disappeared (I). Arrowheads represent dead granulosa cells; arrows represent fibroblast cells; short arrows represent intumescent cells. T, theca layer; TI, theca interna layers; TE, theca externa layers; ZP, zona pellucida. Bar = 20 m.
and their shapes changed from slender to intumescent. Subsequently, the new tissue formed and gradually became the dominant part in the antrum (Fig. 1F). In addition, the volume of the atretic follicle shrunk obviously. However, this new tissue appeared to be a temporary structure during follicular atresia, and degenerated
in a manner similar to hyalinization (Fig. 1G). Moreover, the disappearance of the new tissue did not mean the end of atresia, as loose connective tissue with abundant ECM appeared again in the follicle (Fig. 1H). With further shrinking of follicular volume, the loose connective tissues gradually lessened, and the oocyte degen-
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Fig. 3. Immunohistochemical localization of PCNA in follicular atresia. Ovarian sections were prepared for immunohistochemistry and PCNA immunoreactivity was detected using a monoclonal antibody against the PCNA. Positive immunostaining for PCNA is indicated by a brown reaction product. In healthy follicles, granulosa cells were strongly stained, and theca cells also stained positively. However, granulosa cells and theca cells did not stain after the elimination of granulosa cells (A and B). During fibroblast-like cell invaded into the antrum to form new tissue, the cells were positively stained (C and D). When the new tissue degenerated, it still stained positively (E). In control experiments, no significant immunoreactivity was observed when normal bovine serum (BSA) was used instead of primary antibody (F). H, healthy follicles; AF, atretic follicles; NT, new tissue in atretic follicles. Bar = 50 m.
erated entirely and the zona pellucida showed irregular shapes. At this stage, the theca layers became compact, and a small cavity was formed in the follicle, which was enveloped by fibroblast-like cells (Fig. 2H). Finally, the atretic follicle shrank into a small volume, and the boundary between follicular cells and stromal cells became indistinguishable (Fig. 1I and Fig. 2I). 3.3. Cellular morphologic changes in theca interna layers The morphologic studies on theca interna cells showed that granulosa layers were eliminated rapidly in the process of follicular atresia (Fig. 2); however, the theca cells underwent a
series of changes, primarily with regard to cellular shape and orientation. In healthy follicles, theca interna cells were arranged regularly, and had an oval and orderly nucleus (Fig. 2A). However, when the granulosa cells began to undergo apoptosis, the theca cells changed to irregular shapes (Fig. 2B and C). Later, when the granulosa cells were eliminated entirely (Fig. 2D), the theca cells had round shapes, and the fibroblast-like cell appeared in the innermost layer. Moreover, the farther from the oocyte, the more obvious changes observed (Fig. 1C). Subsequently, many fibroblast-like cells appeared in the theca layer, and edged in the follicular antrum (Fig. 2E). At this stage, the connections in the theca layer were very
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Table 1 Classification of atretic follicles. Phases
Granulosa cells (GCs) and theca cells (TCs)
Fibroblast-like cells (FBs)
Cells in the antrum or outside of ZPa
I
None.
None.
None.
None.
III
GCs and TCs arranged neatly, and the TCs have oval shapes. Pyknotic cells appear in GC layer, and the arrangement of TCs is loose. GCs eliminated, and TCs become round.
IV
GCs eliminated almost entirely.
Mass of dead GCs float along the antrum. The antrum center is empty.
V
Shapes of TCs become irregular.
VI
Connections in the TC layers very loose.
VII VIII
TC layers become compact to some extent. TCs have round shapes.
FBs appear in the innermost layer of theca cells. FBs surround the antrum with many layers. Many FBs appear in the TCs, and migrate to the antrum. Massive FBs migrate to the antrum with very long protrusions. Number of FBs decrease.
IX
TC layers become compact.
Several layers of FBs appear outside the ZP, and form a small cavity.
X
The TC layers become very thin.
None.
II
a
Only a few FBs observed.
Loose connective tissue forms in the antrum with FBs and intumescent cells. Many FBs and intumescent cells congregate outside the ZP. The new tissue forms in the antrum with many intumescent cells. New tissue degenerates in a hyalinized manner. New tissue disappears, but the loose connective tissue appears again outside the ZP. Loose connective tissue disappears.
ZP, zona pellucida.
Fig. 4. Measurements of follicular diameter (A), thickness of theca interna cells (B), and the ratio of L1 (diameter in centripetal direction) to L2 (diameter in tangent direction) in nuclei of theca interna cells (C). Each value represents the mean ± SEM. One-way analysis of variance (ANOVA) was used to analyze the data. Differing superscripts a–h denote significant values (p < 0.05) by Duncan’s multiple-range test, while the same letters denote lack of significance (p > 0.05).
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loose, and the leading edge of the fibroblast-like cells extended as very long protrusions to the antrum (Fig. 2F). Meanwhile, the theca interna cells became compact and showed irregular shapes (Fig. 2G). Then fibroblast-like cells appeared in the follicle, and formed a small cavity outside the zona pellucida (Fig. 2H). Finally, with the shrunken volume of follicles, fibroblast-like cells disappeared (Fig. 4F). 3.4. Cell proliferation in the atretic follicles Using PCNA as a marker of cell proliferation, the results of immunohistochemical staining revealed that the cells proliferated during the process of follicular atresia (Fig. 3). In healthy follicles, granulosa cells were strongly stained, and theca cells also stained positively. However, granulosa cells and theca cells did not stain after the elimination of granulosa cells (Fig. 3A and B). During fibroblast-like cell invaded into the antrum to form new tissue, the cells were positively stained (Fig. 3C and D). When the new tissue degenerated, it still stained positively (Fig. 3E).
in distinct contrast to the situation in the other species mentioned, where the antrum is always empty during the entire process of atresia. Surprisingly, the new tissue degenerated by a hyalinization process, as occurs in follicular walls in the bovine (Marion et al., 1968), yaks (Cui and Yu, 1999) and dog (Spanel-Borowski, 1981). The new tissue was formed in the follicular antrum, and this process was similar to wound healing in some respects (Bullard et al., 2003; Huttenlocher et al., 1995; Li et al., 2007; Martin, 1997). We hypothesize that, after the elimination of granulosa cells, the formation of new tissue might be a central event in the process of follicular atresia. Although little is known about this new tissue at present, it might be functional. This requires further investigation. In addition, the oocyte and zona pellucida may be influential in the process of follicular atresia. After the elimination of granulosa cells, we found that cells close to the oocyte and zona pellucida appeared to be inactive, while those more distal were apparently quite active. Although it was reported that the oocyte might prevent granulosa cell differentiation (Vanderhyden, 1996), it is currently unknown as to how the oocyte performs this function and the signaling pathways involved. This requires further future study.
3.5. Evaluation of follicular atresia 5. Conclusions Based on the cellular changes described above, the process of follicle atresia was divided into 10 sequential phases, and a Roman numeral I to X was assigned to each phase (Table 1). Relative to this 10-phase classification, the follicular diameters, thickness of theca interna cells, and the ratio of L1 to L2 in theca interna cell nuclei were measured to evaluate morphologic changes (Fig. 4). There were significant differences among different phases, except among phases VI, VII, VIII (Fig. 4A). It was indicated that the follicles shrank during the process of follicular atresia. However, the measurements of theca interna cells were more complex (Fig. 4B). The minimal value appeared in phases I and II, and significantly increased twice during the process of follicular atresia. The first peak value appeared in phases IV and V, and the second appeared in phase IX. Because the loose connective tissue appeared in the same phases (phases V and IX), there might be some correlation between them. The ratio of L1/L2 in theca interna cell nuclei indicated changes in cellular arrangement (Fig. 4C), and the value 1.0 refers to cells with spherical shape. It was evident that the arrangement of theca cells changed twice during the process of follicular atresia. Moreover, there were no significant differences between phases VII and VIII regarding any measurements (Fig. 4). Surprisingly, the loose connective tissue appeared both before and after the formation of the new tissue, and the corresponding phases were V and IX. It is suggested that the loose connective tissue played a very important role in the formation and degeneration of new tissue. 4. Discussion In the current study, we have described the morphological changes in detail during the process of follicular atresia in guinea pigs, and demonstrated cell proliferation coexisted. After the elimination of granulosa cells, dramatic changes occurred in the theca layers, and loose connective tissue and new tissue appeared outside the zona pellucida. It is suggested that the process of follicular atresia is precise, and that atretic follicles are vigorous structures. Compared with other species, such as mice (Byskov, 1974), rats (Osman, 1985; Tabarowski et al., 2005), goats (Garcia et al., 1997), dogs (Spanel-Borowski, 1981), yaks (Cui and Yu, 1999) and bovine (Marion et al., 1968), there is little evidence that confirms cell proliferation existing in atretic follicles in guinea pigs. It was indicated that the manner of follicular atresia was obviously different for guinea pigs. With loose connective tissue and new tissue appearing in guinea pigs, the antrum was occupied substantially. This is
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Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Involvement of cell proliferation in the process of follicular atresia in the guinea pig Wei Wang a , Honglin Liu a , Wei Ding a,b , Yan Gong a , Jingwei Chen a , Reinhold J. Hutz c , Dagan Mao a , Fangxiong Shi a,∗ a b c
Laboratory of Animal Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China Department of Animal Husbandry and Veterinary Medicine, Jiangsu Polytechnic College of Agriculture and Forestry, Jurong 212400, PR China Department of Biological Sciences, University of Wisconsin-Milwaukee, WI 53201, USA
a r t i c l e
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Article history: Received 20 January 2010 Received in revised form 21 April 2010 Accepted 27 April 2010 Available online 3 June 2010 Keywords: Follicular atresia Cell proliferation Guinea pig
a b s t r a c t Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction A considerable amount of research has been performed on follicular atresia during the last decade, and the main work focused on cell death and elimination (Alonso-Pozos et al., 2003; Nezis et al., 2006; Santos et al., 2008). However, little was known about the cell proliferation in the process of follicular atresia. In our current paper, these phenomena were documented in detail in guinea pigs. In view of some morphologic characteristics described for the first time, several special terms were cited based on the similarity of structures and functions. In the process of follicular atresia, the morphologic changes were obvious, and some polymorphic cells are differentiated from the innermost theca layer, which were described as “stellate or slender cells” (Kasuya, 1997), “fibroblastoid cells”, “fibroblast-like cells” and “fibroblastoid-looking cells” (Logothetopoulos et al., 1995). In the present paper, the polymorphic cells were denominated as “fibroblast-like cells” (Wang et al., 2010).
∗ Corresponding author. E-mail addresses: [email protected] (W. Wang), [email protected] (F. Shi). 0040-8166/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2010.04.006
It had been reported that a large numbers of fibroblast-like cells appeared in the antrum of atretic follicles, and documented as a “cellular network” (Kasuya, 1997). However, cell proliferation was not confirmed (Kasuya, 1997; Logothetopoulos et al., 1995). In the present study, the protein proliferating cell nuclear antigen (PCNA) was used as the proliferation marker (Bolton et al., 1994; Paunesku et al., 2001; Wildemann et al., 2003). PCNA – a cofactor of DNA polymerases that encircles DNA – orchestrates several of these functions by recruiting crucial players to the replication fork (Moldovan et al., 2007). Our results showed that cell proliferation did occur in atretic follicles. In addition, the term “cellular network” was not included in the changes within the follicular antrum. When a large number of fibroblast-like cells appeared, the antrum was not empty any more, and had very loose connections. This structure was described as “loose connective tissue” (Reed et al., 2009; Tzouvelekis et al., 2009; Weber, 1999; Wolf et al., 2009). Subsequently, with the proliferation of the differentiated cells, a new tissue formed in the antrum, which was described as “new tissue” (Leite et al., 2002). Although obvious changes occurred in the theca layers, the follicles were destined to degenerate. With the shrinking of follicular volume, atretic follicles lost their identifications (Logothetopoulos et al., 1995). Cellular morphologic changes during follicular atresia were fully investigated in the present study using histology, and we confirmed
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follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation.
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Twenty-five adult female guinea pigs (Cavia porcellus) of the Hartley strain were used at 5 months of age, with an initial weight of 400–700 g. They were housed 4 animals per cage, under controlled temperature at 23 ± 2 ◦ C, and fed commercial food and tap water ad libitum. Estrous cycles were recorded by daily examination of vaginal smears whenever the vagina was open (Shi et al., 1999). The day of ovulation was estimated as the day when the maximal cornification was seen in the smear (Hutz et al., 1990; Lilley et al., 1997; Norris and Adams, 1979) and was designated as day 0 of the cycle. Days of the estrous cycle followed thereafter consecutively. We used only animals showing at least two consecutive 15–17-day cycles immediately prior to the experiment. The animals were sacrificed on days 1, 4, 8, 12, and 16 (5 animals per day) (Garris and Mitchell, 1979). Then the ovaries were collected immediately for experiments.
of L1 (diameters in centripetal direction) to L2 (diameters in tangent direction) in the nuclei of theca interna cells were measured for evaluating the follicular atresia. It was widely accepted that the follicular growth of guinea pigs was biphasic (Bland, 1980; Fortune, 1994; Hutz et al., 1990; Shi et al., 1999). In the current study, follicles on days 16, 1 and 4 were investigated, which constituted the second wave of follicular growth and atresia. Only those follicles with the largest cross-sectional area were measured, and we ensured that each follicle was measured only once. The sample size of each group was at least twenty. Microscopic measurements were made with a Nikon 80i (Nikon, Tokyo, Japan). Follicular diameter was taken as the mean of the largest diameter and the diameter perpendicular to the midpoint (Bland, 1980). Thickness of theca interna cells was measured at three different positions, and the mean was used for statistical analyses. The ratio of L1 to L2 was taken to denote the arrangement changes in the nuclei of theca interna cells, and the migrated fibroblast-like cells which had very thin shapes were excluded. At least three areas at 50 m × 50 m were measured in each follicle, and the mean was used for statistical analyses. Therefore, the ratio more than or less than 1.0, respectively, represented the arrangement of theca interna cells in centripetal or tangent direction.
2.2. Reagents
3. Results
PCNA monoclonal antibodies were purchased from Biogenix Co. (San Ramon, CA, USA, lot: Mu2060899); immunohistochemical kits (SABC methods) were purchased from Boshide Co. (Wuhan, Hubei, China, lot: SA1021); 3,3 -diaminobenzidine tetrachloride (DAB) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were purchased reagent grade.
3.1. Follicular atresia in normal estrous cycles
2. Materials and methods 2.1. Animals and sample collection
2.3. Histological and immunohistological studies Ovaries were fixed in 4% paraformaldehyde at room temperature for 24 h, embedded in paraffin, sectioned serially at 10 m and stained with hematoxylin and eosin (HE). These sections were analyzed for the morphologic changes indicative of atretic follicles. To examine the proliferating cells in the atretic follicles, immunohistochemical staining was performed using monoclonal antibodies to PCNA with the streptavidin–biotin-complex (SABC) method. A few sections were picked randomly from the serial sections. Sections were mounted on slides coated with APES and which were dried for 24 h at 37 ◦ C. The antibodies were diluted 1:500 in PBS containing 1% bovine serum albumin (BSA), and the sections were incubated overnight at 4 ◦ C with primary antibodies. The specific protein immunoreactivity was visualized by 0.05% DAB in 10 mM PBS-buffered saline containing 0.01% H2 O2 for 2–5 min, and counterstained with hematoxylin. A negative control was a primary antibody blank, and relative levels of immunostaining were evaluated and repeated at least four times. The percent area of follicular cells staining positive for PCNA by more than 50% were considered strongly positive, between 10 and 50% were considered positive, and less than 10% were considered to be negative. The images were captured in a Nikon 80i (Nikon, Tokyo, Japan). Three areas at 50 m × 50 m were selected randomly for evaluation within each follicle, and at least six follicles were evaluated for each stage of follicular atresia. 2.4. Evaluation of follicular atresia Follicles were considered as round shape in the sections, and cellular arrangement was assigned in centripetal or tangent direction. The tangent direction was perpendicular to centripetal direction. Follicular diameter, thickness of theca interna cells, and the ratio
On day 1 after ovulation, there were very few healthy antral follicles in the ovaries, and the new corpora lutea were observed. A mass of dead granulosa cells separated from the granulosa layers and scattered in the antrum. Furthermore, in some atretic follicles where granulosa cells were eliminated, fibroblast-like cells appeared in the theca interna cells. On day 4, the most atretic follicles were shrunken, and the follicular antrum was filled with fibroblast-like cells. Some atretic follicles exhibited a very small volume and the fibroblast-like cells disappeared. On days 8 and 12, a group of large antral follicles appeared. Many of these acquired large population of granulosa cells. However, the antral follicles were often in early stages of atresia. On day 16, antral follicles were further developed. Ovaries at this time contained 0–3 unruptured preovulatory follicles, which had diameters at least 700 m. Although many follicles showed large volumes, there was a mass of desquamated granulosa cells and were distributed along the follicular cavity. At this time, it was obvious that the intensity of the follicular atresia was greater than on either day 8 or 12. 3.2. Cell elimination in follicular atresia Based on the morphologic observations (Figs. 1 and 2), there were continuous changes in the process of follicle atresia in guinea pigs. The most remarkable characteristics included the elimination of granulosa cells and the formation of new tissue. Moreover, it seemed that the fibroblast-like cells played a central role in new tissue formation. In healthy follicles, granulosa cells were arranged neatly (Fig. 1A and Fig. 2A). When the atretic process began, connections in the granulosa layers became loose, and the columnar granulosa cells became round (Fig. 2B), with many pyknotic cells and apoptosis bodies were observed (Fig. 2C). With the massive elimination of granulosa cells, granulosa layers disintegrated gradually, and dead granulosa cells were shed into the follicular antrum (Fig. 1B). At the same time, the fibroblast-like cells appeared in the innermost layer of theca cells (Fig. 2D).
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Fig. 1. Morphologic observations on follicular atresia. In healthy follicles, granulosa cells were arranged neatly (A). With the granulosa cells eliminated (B), the fibroblast-like cells appeared and invaded the antrum (C), and formed a type of loose connective tissue (D). Meanwhile, more and more cells congregated outside the zona pellucida (E), and formed a new tissue in the antrum (F). Subsequently, the new tissue degenerated in a manner similar to hyalinization (G), and the loose connective tissue appeared again (H). Finally, the follicle exhibited a very small volume (I). Arrowheads represent dead granulosa cells; arrows represent fibroblast cells. O, oocyte; ZP, zona pellucida; G, granulosa layer; T, theca layer; A, follicular antrum; LCT, loose connective tissue; NT, new tissue. Bar = 50 m.
After the complete elimination of granulosa cells, the innermost layer of theca cells had fibroblast-like shapes (Fig. 1C and Fig. 2E). Later, the fibroblast-like cells appeared in the follicular antrum solely, and the leading edge extended very long protrusions to the antrum (Fig. 2F). Subsequently, a kind of loose connective tissue invaded the whole antrum subsequently. The antrum was then no
longer empty at that period, but filled with many cells and ECM (Fig. 1D). Call–Exner bodies sometimes appeared in the antrum, while the cells close to the antrum were arranged in a centripetal orientation (Fig. 1E). With more and more fibroblast-like cells appeared in the antrum, a mass of cells congregated surrounded the zona pellucida,
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Fig. 2. Morphologic observations on theca interna cells. In healthy follicles, the theca interna cells manifested an orderly nucleus, and were arranged in the same orientation (A). With the process of atresia (B and C), dead granulosa cells were shed into the follicle antrum, and fibroblast-like cells appeared in the innermost layer of theca cells (D). Later, many fibroblast-like cells appeared in the theca layer (E) and the antrum (F). When fibroblast-like cells were few in the theca layer, some intumescent cells appeared in the antrum (G). Subsequently, the follicular volume shrank, and the fibroblast-like cells appeared again (H). Finally, only a few layers of theca interna cells remained, and the fibroblast-like cells disappeared (I). Arrowheads represent dead granulosa cells; arrows represent fibroblast cells; short arrows represent intumescent cells. T, theca layer; TI, theca interna layers; TE, theca externa layers; ZP, zona pellucida. Bar = 20 m.
and their shapes changed from slender to intumescent. Subsequently, the new tissue formed and gradually became the dominant part in the antrum (Fig. 1F). In addition, the volume of the atretic follicle shrunk obviously. However, this new tissue appeared to be a temporary structure during follicular atresia, and degenerated
in a manner similar to hyalinization (Fig. 1G). Moreover, the disappearance of the new tissue did not mean the end of atresia, as loose connective tissue with abundant ECM appeared again in the follicle (Fig. 1H). With further shrinking of follicular volume, the loose connective tissues gradually lessened, and the oocyte degen-
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Fig. 3. Immunohistochemical localization of PCNA in follicular atresia. Ovarian sections were prepared for immunohistochemistry and PCNA immunoreactivity was detected using a monoclonal antibody against the PCNA. Positive immunostaining for PCNA is indicated by a brown reaction product. In healthy follicles, granulosa cells were strongly stained, and theca cells also stained positively. However, granulosa cells and theca cells did not stain after the elimination of granulosa cells (A and B). During fibroblast-like cell invaded into the antrum to form new tissue, the cells were positively stained (C and D). When the new tissue degenerated, it still stained positively (E). In control experiments, no significant immunoreactivity was observed when normal bovine serum (BSA) was used instead of primary antibody (F). H, healthy follicles; AF, atretic follicles; NT, new tissue in atretic follicles. Bar = 50 m.
erated entirely and the zona pellucida showed irregular shapes. At this stage, the theca layers became compact, and a small cavity was formed in the follicle, which was enveloped by fibroblast-like cells (Fig. 2H). Finally, the atretic follicle shrank into a small volume, and the boundary between follicular cells and stromal cells became indistinguishable (Fig. 1I and Fig. 2I). 3.3. Cellular morphologic changes in theca interna layers The morphologic studies on theca interna cells showed that granulosa layers were eliminated rapidly in the process of follicular atresia (Fig. 2); however, the theca cells underwent a
series of changes, primarily with regard to cellular shape and orientation. In healthy follicles, theca interna cells were arranged regularly, and had an oval and orderly nucleus (Fig. 2A). However, when the granulosa cells began to undergo apoptosis, the theca cells changed to irregular shapes (Fig. 2B and C). Later, when the granulosa cells were eliminated entirely (Fig. 2D), the theca cells had round shapes, and the fibroblast-like cell appeared in the innermost layer. Moreover, the farther from the oocyte, the more obvious changes observed (Fig. 1C). Subsequently, many fibroblast-like cells appeared in the theca layer, and edged in the follicular antrum (Fig. 2E). At this stage, the connections in the theca layer were very
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Table 1 Classification of atretic follicles. Phases
Granulosa cells (GCs) and theca cells (TCs)
Fibroblast-like cells (FBs)
Cells in the antrum or outside of ZPa
I
None.
None.
None.
None.
III
GCs and TCs arranged neatly, and the TCs have oval shapes. Pyknotic cells appear in GC layer, and the arrangement of TCs is loose. GCs eliminated, and TCs become round.
IV
GCs eliminated almost entirely.
Mass of dead GCs float along the antrum. The antrum center is empty.
V
Shapes of TCs become irregular.
VI
Connections in the TC layers very loose.
VII VIII
TC layers become compact to some extent. TCs have round shapes.
FBs appear in the innermost layer of theca cells. FBs surround the antrum with many layers. Many FBs appear in the TCs, and migrate to the antrum. Massive FBs migrate to the antrum with very long protrusions. Number of FBs decrease.
IX
TC layers become compact.
Several layers of FBs appear outside the ZP, and form a small cavity.
X
The TC layers become very thin.
None.
II
a
Only a few FBs observed.
Loose connective tissue forms in the antrum with FBs and intumescent cells. Many FBs and intumescent cells congregate outside the ZP. The new tissue forms in the antrum with many intumescent cells. New tissue degenerates in a hyalinized manner. New tissue disappears, but the loose connective tissue appears again outside the ZP. Loose connective tissue disappears.
ZP, zona pellucida.
Fig. 4. Measurements of follicular diameter (A), thickness of theca interna cells (B), and the ratio of L1 (diameter in centripetal direction) to L2 (diameter in tangent direction) in nuclei of theca interna cells (C). Each value represents the mean ± SEM. One-way analysis of variance (ANOVA) was used to analyze the data. Differing superscripts a–h denote significant values (p < 0.05) by Duncan’s multiple-range test, while the same letters denote lack of significance (p > 0.05).
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loose, and the leading edge of the fibroblast-like cells extended as very long protrusions to the antrum (Fig. 2F). Meanwhile, the theca interna cells became compact and showed irregular shapes (Fig. 2G). Then fibroblast-like cells appeared in the follicle, and formed a small cavity outside the zona pellucida (Fig. 2H). Finally, with the shrunken volume of follicles, fibroblast-like cells disappeared (Fig. 4F). 3.4. Cell proliferation in the atretic follicles Using PCNA as a marker of cell proliferation, the results of immunohistochemical staining revealed that the cells proliferated during the process of follicular atresia (Fig. 3). In healthy follicles, granulosa cells were strongly stained, and theca cells also stained positively. However, granulosa cells and theca cells did not stain after the elimination of granulosa cells (Fig. 3A and B). During fibroblast-like cell invaded into the antrum to form new tissue, the cells were positively stained (Fig. 3C and D). When the new tissue degenerated, it still stained positively (Fig. 3E).
in distinct contrast to the situation in the other species mentioned, where the antrum is always empty during the entire process of atresia. Surprisingly, the new tissue degenerated by a hyalinization process, as occurs in follicular walls in the bovine (Marion et al., 1968), yaks (Cui and Yu, 1999) and dog (Spanel-Borowski, 1981). The new tissue was formed in the follicular antrum, and this process was similar to wound healing in some respects (Bullard et al., 2003; Huttenlocher et al., 1995; Li et al., 2007; Martin, 1997). We hypothesize that, after the elimination of granulosa cells, the formation of new tissue might be a central event in the process of follicular atresia. Although little is known about this new tissue at present, it might be functional. This requires further investigation. In addition, the oocyte and zona pellucida may be influential in the process of follicular atresia. After the elimination of granulosa cells, we found that cells close to the oocyte and zona pellucida appeared to be inactive, while those more distal were apparently quite active. Although it was reported that the oocyte might prevent granulosa cell differentiation (Vanderhyden, 1996), it is currently unknown as to how the oocyte performs this function and the signaling pathways involved. This requires further future study.
3.5. Evaluation of follicular atresia 5. Conclusions Based on the cellular changes described above, the process of follicle atresia was divided into 10 sequential phases, and a Roman numeral I to X was assigned to each phase (Table 1). Relative to this 10-phase classification, the follicular diameters, thickness of theca interna cells, and the ratio of L1 to L2 in theca interna cell nuclei were measured to evaluate morphologic changes (Fig. 4). There were significant differences among different phases, except among phases VI, VII, VIII (Fig. 4A). It was indicated that the follicles shrank during the process of follicular atresia. However, the measurements of theca interna cells were more complex (Fig. 4B). The minimal value appeared in phases I and II, and significantly increased twice during the process of follicular atresia. The first peak value appeared in phases IV and V, and the second appeared in phase IX. Because the loose connective tissue appeared in the same phases (phases V and IX), there might be some correlation between them. The ratio of L1/L2 in theca interna cell nuclei indicated changes in cellular arrangement (Fig. 4C), and the value 1.0 refers to cells with spherical shape. It was evident that the arrangement of theca cells changed twice during the process of follicular atresia. Moreover, there were no significant differences between phases VII and VIII regarding any measurements (Fig. 4). Surprisingly, the loose connective tissue appeared both before and after the formation of the new tissue, and the corresponding phases were V and IX. It is suggested that the loose connective tissue played a very important role in the formation and degeneration of new tissue. 4. Discussion In the current study, we have described the morphological changes in detail during the process of follicular atresia in guinea pigs, and demonstrated cell proliferation coexisted. After the elimination of granulosa cells, dramatic changes occurred in the theca layers, and loose connective tissue and new tissue appeared outside the zona pellucida. It is suggested that the process of follicular atresia is precise, and that atretic follicles are vigorous structures. Compared with other species, such as mice (Byskov, 1974), rats (Osman, 1985; Tabarowski et al., 2005), goats (Garcia et al., 1997), dogs (Spanel-Borowski, 1981), yaks (Cui and Yu, 1999) and bovine (Marion et al., 1968), there is little evidence that confirms cell proliferation existing in atretic follicles in guinea pigs. It was indicated that the manner of follicular atresia was obviously different for guinea pigs. With loose connective tissue and new tissue appearing in guinea pigs, the antrum was occupied substantially. This is
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