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Potential roles of matrix metalloproteinases and characteristics of ovarian development in neonatal guinea pigs
Tissue and Cell 47 (2015) 478–488
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Potential roles of matrix metalloproteinases and characteristics of ovarian development in neonatal guinea pigs Junrong Li a,b , Ting Shen b , Guoyun Wu a , Quanwei Wei a , Dagan Mao a , Fangxiong Shi a,∗ a b
Laboratory of Animal Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China College of Agriculture and Bio-engineering, Jinhua Polytechnic, Jinhua 321017, China
a r t i c l e
i n f o
Article history: Received 27 May 2015 Received in revised form 1 July 2015 Accepted 20 July 2015 Available online 21 July 2015 Keywords: eCG Neonatal guinea pigs StAR PCNA MMP-2 MMP-9
a b s t r a c t The present study was conducted to investigate expression of matrix metalloproteinases (MMPs) and early ovarian development in neonatal guinea pigs. Thirty neonatal guinea pigs at 3 or 8 days of age were administrated 5 IU equine chorionic gonadotropin (eCG) or saline, and the ovaries were collected after 2 days of eCG. Serum concentrations of estradiol and progesterone were determined, and ovarian localization of StAR, MMP-2 and MMP-9 were analyzed by immunohistochemical staining. Results indicate that injection of eCG sensitized the neonatal ovary and elevated serum concentrations of estradiol and progesterone, but not enough to stimulate ovarian follicular development in the ovaries. MMP-2 and MMP-9 were both immunolocalized to the surface of granulosa cells of primary and secondary follicles, and high MMP-2 expression was accompanied by low StAR expression in eCG-treated ovaries. Collectively, we hypothesize that MMP-2, -9 and StAR are both involved in follicular atresia through their participation in cell proliferation and tissue remodeling. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Guinea pigs share some similarities in their reproductive cycle with humans, have a small ovulatory quota, and constitute a reliable animal model for studying human reproduction (Kulduk et al., 2014). However, ovarian development after birth in guinea pigs has been studied rarely, when compared with other rodents. Gonadotropins are basically used to induce multiple ovulations in animals, and equine chorionic gonadotropin (eCG) is a standard gonadotropin used to recruit additional follicles (Bó and Mapletoft, 2014). eCG plays a role similar to that of follicle-stimulating hormone (FSH) in superovulation (Mapletoft et al., 1990), but has a half-life up to 40 h (Zanetti et al., 2014; Murphy and Martinuk, 1991). eCG was effective in rats and mice, but eCG did not stimulate superovulation in adult guinea pigs (Suzuki et al., 2003). Our previous study showed that eCG played a role in adult guinea pigs similar to that of luteinizing hormone (LH); i.e., eCG induced luteinized unruptured follicle (LUF) syndrome in cyclic guinea pigs (Supplement Material). Our understanding of the sensitivity of
∗ Corresponding author. E-mail address: [email protected] (F. Shi). http://dx.doi.org/10.1016/j.tice.2015.07.005 0040-8166/© 2015 Elsevier Ltd. All rights reserved.
neonatal guinea pigs to eCG is lacking and the etiology of LUFs is still unknown. Matrix metalloproteinases (MMPs), which participate in tissue remodeling (Kessenbrock et al., 2010), are also involved in morphogenesis, apoptosis, ovulation and angiogenesis (Jones, 2014; Roy et al., 2006; Vu and Werb, 2000). MMPs play a critical role in follicular development in ovaries by remodeling the extracellular matrix (ECM) in the periovulatory period (Bagavandoss, 1998; Baker et al., 2000; Cooke et al., 1999; McCaffery et al., 2000). MMP-2 and MMP-9, both with a fibronectin-like domain in the middle of the catalytic domain (Bode et al., 1999), can degrade collagens, and possess laminin and aggrecan core proteins (Nagase et al., 2006), and MMP-2 and MMP-9 were both immunolocalized in developing follicular theca and stroma in rodents (Garcia et al., 1997). The intrafollicular levels of MMP-2 and MMP-9 both increased during the progression from normal to atresia ovarian follicles in sheep (Huet et al., 1997). A higher production of MMP-2 and MMP-9 was also accompanied by a higher follicular apoptosis rate in patients (Ben-Shlomo et al., 2003; Shalev et al., 2001). The objective of the present study was to investigate the role of eCG in neonatal guinea pigs, with a focus on expression of MMP-2 and MMP-9 in ovaries. In addition, immunolocalization of steroidogenic acute regulatory protein (StAR) and
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proliferating cell nuclear antigen (PCNA) were performed, since they are the indicators of steroidogenesis (Clark et al., 1997) and cell proliferation (Sun et al., 2014; Wildemann et al., 2003), respectively.
2. Materials and methods Thirty neonatal female Harley-White guinea pigs (Cavia porcellus) at 3 days of age were obtained from the Laboratory Animal Research Center of Zhejiang Chinese Medical University (Hangzhou, China). The guinea pigs were randomly divided into 5 groups (each n = 6): groups D-3, D-5, P-5, D-10 and P-10. The animals in group D-3 were sacrificed at 3 days of age; the animals in groups P-5 and D-5 were administered 5 IU eCG or saline at 3 days of age, and then sacrificed at 5 days; the animals in groups P-10 and D-10 were administered 5 IU eCG or saline at 8 days of age, respectively, and then sacrificed at 10 days of age. Blood samples were collected before scarification; and ovarian samples were collected immediately after scarification. The serum was separated from blood by centrifugation at 5000 g for 10 min, and the serum concentrations of estradiol and progesterone were measured (Adicon Clinical Laboratories INC, Hangzhou, China). The ovaries were fixed in 4% paraformaldehyde at room temperature for 36 h, and then kept in 70% alcohol for histologic and immunohistochemical (IHC) analyses. After fixation, ovarian samples were embedded in paraffin, sectioned serially at 5 m and stained with H&E. Sections were analyzed for observations of morphologic changes in ovaries after eCG injection. The follicular stages in neonatal guinea pigs were determined according to a previous study on mouse ovaries (Flaws et al., 1997). The numbers of primordial and antral follicles were both counted: primordial follicles in guinea pigs contained only one layer of granulosa cells, while antral follicles contained an antrum. In order to immunolocalized steroidogenesis in ovarian follicles, we performed IHC staining using monoclonal antibodies against StAR and PCNA (Santa Cruz Biotechnology Inc., TX, USA; SC25806, lot: I2308; SC7907, lot: L5346) with a strept avidin-biotin complex (SABC) kit (Boshide Biotechnology Inc., Wuhan, China; SA2002, lot: 10C09A). Ovarian expressions of MMP-2 and MMP9 were characterized by IHC staining with antibodies to MMP-2 and MMP-9 with an SABC kit (all from Fuzhou Maixin Biotech Co., Ltd., Fuzhou, China; lot: MAB-0244; lot: MAB-0245; lot: KIT5910). The antibodies were diluted to 1:200 in phosphate-buffer saline (PBS) containing 1% bovine serum albumin (BSA). Heatinduced epitope retrieval (HIER) was performed by heating the slides immersed in the 10 mM sodium citrate buffer (pH 6.0) at 100 ◦ C in a microwave oven for 8 min. The sections were mounted on slides coated with 3-aminopropyl-triethoxysilane (APES), and dried at 37 ◦ C for 24 h. The sections were incubated with the primary antibodies overnight at 4 ◦ C. The immunoreactivity was visualized using diaminobenzidine (DAB) (Sigma-Aldrich Corp., MO, USA) as substrate and counter-stained with hematoxylin. Normal rabbit serum, instead of primary antibody, was used as negative control. The stained ovarian samples were observed in a BX51 electrical microscope (Olympus Corporation, Tokyo, Japan). Images were recorded by an XC10 optical imaging system (Olympus Corporation), and analyzed on OlyVIA (version 2.4, Olympus Soft Imaging Solutions GmbH, Japan). Statistical analyses were performed on IBM SPSS Statistics 21 (Chicago, IL, USA). Differences were evaluated by one-way analysis of variance (ANOVA) with followed by Tukey’s test. P < 0.05 was considered to be significant.
Fig. 1. Serum concentrations of estradiol and progesterone in neonatal guinea pigs after eCG injection. D-5, 5-day-old guinea pigs with saline injection at 3 days of age; P-5: 5-day-old guinea pigs after injection with 5 IU eCG at 3 days of age; D10, 10-day-old guinea pigs with saline injection at 8 days of age; P-10: 10-day-old guinea pigs with injection of 5 IU eCG at 8 days of age. Each value is expressed as mean ± SEM. Different superscripts represent significant differences among categories (P < 0.05) as analyzed by Tukey test; while identical letters denote non significance (P > 0.05). F-test for homogeneity of variance, P > 0.05; W-test for normality of distribution, P > 0.05.
3. Results 3.1. Serum concentrations of estradiol and progesterone in neonatal guinea pigs As shown in Fig. 1, the serum concentrations of estradiol are significantly higher in group P-5 vs. group D-5, in group P-10 vs. group D-10, and in group P-10 vs. group P-5 (all P < 0.05); the serum concentrations of progesterone are significantly higher in group P5 vs. group D-5, in group P-10 vs. group D-10, in group P-10 vs. group P-5, and in group D-10 vs. group D-5 (all P < 0.05). 3.2. Observation of ovarian morphology in neonatal guinea pigs Few antral follicles were observed in the ovaries of group D3 (Fig. 2A). Specifically, primordial follicles were distributed close to ovarian walls (Fig. 2A1) and surrounded primordial follicles.
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Fig. 2. Changes in ovarian morphology in neonatal guinea pigs in D-3, D-5 and D-10 groups using H&E stain. D-3, normal 3-day-old guinea pigs. Oo: oocyte. GC: granulosa cell. TC: theca cell.
Moreover, the oocytes in primary follicles were closely located with granulosa cells (Fig. 2A2), while the secondary follicles were quite small in size (Fig. 2A3). Compared with group D-3, the number of antral follicles in group D-5 was significantly larger (Fig. 2B), the layer of granulosa cells and theca cells showed apparent boundaries (Fig. 2B1), and the secondary follicles were at different developmental stages (Fig. 2B2 and B3). Compared with group D-5, the number of antral follicles in group D-10 was significantly larger (Fig. 2C), the follicular antrum in group D-10 was larger in size (Fig. 2C1 and C2), and antral atretic follicles were observed in group D-10. Moreover, the granulosa cells were separated from
the layer of theca cells, and distributed in the follicular antrum (Fig. 2C3). 3.3. Changes in ovarian morphology in guinea pigs by eCG injection Compared with the ovaries in group D-5 (Fig. 3A), a significantly smaller number of antral follicles was observed in group P-5 (Figs. 3D and 4). The preantral follicles were contiguously arranged in ovaries in group P-5 (Fig. 3E and E1), unlike as in group D-5 where antral follicles were at a similar stage (Fig. 3B
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Fig. 3. Changes in ovarian morphology in neonatal guinea pigs in D-5 and P-5 groups using H&E stain. Oo: oocyte; GC: granulosa cell; TC: theca cell; AF: antral follicle; PmF: primordial follicle.
and B1). Non-antral atretic follicles were observed in both groups D-5 and P-5 (Fig. 3A1 and D1), but the number of primordial follicles in group P-5 was significantly smaller than in group D-5 (Fig. 4). Compared with the ovaries in group D-10 (Fig. 4A), the number of antral follicles in group P-10 was significantly smaller (Fig. 5A). The antral secondary follicles in the ovaries of group D-10 were at different growth stages (Fig. 4B), as indicated by the fact that the layer of granulosa cells was thinner and closely connected to the layer of theca cells (Fig. 4B1). However, the antral follicles in group P-10 were smaller in size vs. in group D-10 (Fig. 4E), and were mostly irregularly-shaped (Fig. 4E1). Compared wtith group D-10, the number of primordial follicles in group P-10 was significantly larger (Fig. 5B) and located in the middle of the ovaries (Fig. 4F1).
Antral atretic follicles were observed in group D-10 (Fig. 4A1), but not in group P-10 (Fig. 4D1). 3.4. Immunolocalization of StAR and PCNA in ovaries from guinea pigs after eCG injection StAR and PCNA were mainly expressed in granulosa cells in small antral secondary follicles of ovaries in group D-3 (Fig. 6A and B). StAR and PCNA were both expressed in antral and nonantral follicles in group D-5, and mainly immunolocalized in layers of granulosa cells in the ovaries (Fig. 6D and E). PCNA was immunolocalized in layers of granulosa cells also, but StAR was mainly expressed in layers of theca cells in primary follicles of ovaries in group P-5 (Fig. 6G and H).
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Fig. 4. Changes in ovarian morphology in neonatal guinea pigs in D-10 and P-10 groups using H&E staining. Oo: oocyte; GC: granulosa cell; TC: theca cell; AF: antral follicle; PmF: primordial follicle.
StAR and PCNA were both expressed in ovaries in group D10, but StAR was barely expressed in granulosa cells separated from theca cells in atretic follicles (Fig. 6J and K). StAR and PCNA were both mildly expressed in ovaries in group P-10, and primarily expressed in granulosa cells of small secondary follicles (Fig. 6M and N). 3.5. Expression of MMP-2 and MMP-9 in ovaries from guinea pigs after eCG injection MMP-2 and MMP-9 were both expressed in ovaries in group D3 (Fig. 7A and B) and D-5 (Fig. 8A and B), and mainly expressed in the layer of granulosa cells in primary and secondary follicles (Figs. 7A1, B1 and 8A1, B1). MMP-2 and MMP-9 were both expressed in ovaries in group P-5 (Fig. 8D and E), and MMP-2 was
also immunolocalized in theca cells of secondary follicles (Fig. 8D1). MMP-2 was expressed in both granulosa cells and theca cells of antral follicles in group P-5 (Table 1), but the expression level of MMP-9 in group P-5 was similar (Table 2), group compared with D-5. MMP-2 and MMP-9 were both immunolocalized in primary and secondary follicles in ovaries of group D-10 (Fig. 9A and B), and mainly expressed in granulosa cells (Fig. 9A1 and B1). MMP-2 and MMP-9 were also expressed in primary and secondary follicles in ovaries of group P-10 (Fig. 9D and E), and both were observed in the layers of granulosa cells (Fig. 9D1 and E1). The MMP-2 and MMP-9 expressions in antral follicles were higher (Table 1), but the MMP-9 expression in primary follicles was lower in group P-10 (Table 2), compared with group D-10.
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Fig. 5. Changes in the numbers of antral and primordial follicles in neonatal guinea pig ovaries after eCG injection. Each value is expressed as mean ± SEM. Different superscripts represent significant differences among categories (P < 0.05) analyzed with the Tukey test; while identical letters denote non significance (P > 0.05). F-test for homogeneity of variance, P > 0.05; W-test for normality of distribution, P > 0.05.
4. Discussion Neonatal guinea pigs at 3 and 8 days of age were sensitive to eCG, as the serum concentrations of progesterone and estradiol were elevated 2 days following injection of 5 IU eCG. The serum progesterone concentration in eCG-treated guinea pigs at 10 days of age was the highest. eCG might play a function similar to LH in
neonatal guinea pigs, which we observed in cyclic adult guinea pigs in our previous study (Li et al., 2015). The antral secondary follicles already appeared in ovaries of 3-day-old guinea pigs, and the follicular development might be initiated in the prenatal period. The ovarian morphology obviously changed during growth of neonatal guinea pigs. Specifically, the number of antral follicles increased steadily at 3, 5
Table 1 Intensity of IHC staining of MMP-2 in ovaries from neonatal guinea pigs after eCG treatment. Cell types
Primordial follicles Oocytes Primary follicles Oocytes GC TC Antral follicles Oocytes GC TC
Control groups
eCG groups
D-3
D-5
D-10
P-5
P-10
+++
+++
+++
++
+++
+++ ++ +/−
+++ ++ +
+++ ++ +
+++ ++ ++
++ ++ ++
+++ +++ +
+++ ++ ++
++ ++ +
++ +++ +++
+++ +++ +++
Note: −, no staining; +/−, very sparse staining; +, faint staining; ++, moderate staining; +++, strong staining.
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Fig. 6. Immunolocalization of StAR and PCNA from ovaries of neonatal guinea pigs. NC: negative control.
and 10 days age; while the percentage of primordial follicles was reduced. In contrast to normal ovaries in 5-day-old guinea pigs, antral follicles were not observed in the ovaries treated at 3 days of age, while the number of primary follicles increased. The results indicated that eCG might inhibit the development from primary to secondary follicles, but promote the recruitment of primary follicles in neonatal ovaries. No large antral secondary follicles were observed in ovaries after eCG injection at day 8. The number of antral follicles was
also lower than in normal 10-day-old ovaries. The number of primary follicles greatly increased in eCG-treated ovaries and the primary follicles were evenly distributed in the middle portions of the ovaries. This indicated that eCG did not stimulate follicular development like FSH, since the antral follicles existing in eCGtreated ovaries might appear even before administration. StAR and PCNA were both expressed in ovaries of 3-day-old guinea pigs, while cellular proliferation of granulosa cells in primary follicles was initiated and accompanied development of
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Fig. 7. Immunolocalization of MMP-2 and MMP-9 in ovaries from neonatal guinea pigs in D-3 group.
primary follicles in neonatal ovaries (Clark et al., 1997). This process might require steroidogenic support (Wildemann et al., 2003). StAR was scarcely expressed in preantral primary follicles of 5day-old ovaries after stimulation with 5 IU eCG. eCG might inhibit steroidogenesis in granulosa cells, and then the development from preantral to antral follicles, which is a potential reason for the absence of antral follicles in treated ovaries. StAR and PCNA were both expressed in atretic follicles in normal 10-day-old ovaries, which indicated that StAR and PCNA were both involved in the atretic process (Xu et al., 2011; Wang et al., 2010). StAR was moderately expressed in ovaries after eCG injection at day 8, which might be a reason for the absence of large antral follicles in eCG-treated ovaries. Thus, eCG might inhibit follicular development and even the activity of FSH in ovaries. MMP-2 and MMP-9 were both expressed in primary and secondary follicles in normal ovaries of 5- or 10-day-old guinea pigs. This result indicated that both MMP-2 and MMP-9 were involved in follicular development in ovaries through their participation in cell proliferation and tissue remodeling (Sternlicht and Werb,
2001; Huet et al., 1997). Both MMPs were immunolocalized on the surface of granulosa cells, and might be involved in regulation of extracellular granulosa cells (Sternlicht et al., 2000). MMP-2 was strongly expressed in eCG-treated ovaries at 3 days of age, and might be highly sensitive to eCG or played a prominent role in tissue remodeling in eCG-treated ovaries. Meanwhile, the high MMP-2 expression was accompanied by the low StAR expression in eCG-treated ovaries, which indicated that the tissue remodeling in ovaries might not depend on steroidogenesis. Compared with normal 10-day-old ovaries, the expression of MMP-9 in primary follicles was reduced in eCG-treated ovaries at 8 days of age, which may be a reason for the small number of antral follicles, and indicated that MMP-2 might be more involved in tissue remodeling in treated ovaries. In conclusion, neonatal ovaries in guinea pigs are already sensitive to the eCG, but follicular development in ovaries was not stimulated by eCG. MMP-2, -9 and StAR might be jointly involved in follicular atresia through their participation in cell proliferation and tissue remodeling.
Table 2 Intensity of IHC staining in MMP-9 in ovaries from neonatal guinea pigs after eCG treatment. Cell types
Primordial follicles Oocytes Primary follicles Oocytes GC TC Antral follicles Oocytes GC TC
Control groups
eCG groups
D-3
D-5
D-10
P-5
P-10
++
+++
+++
+++
+++
++ ++ +/−
+++ + +/−
+ +++ ++
+++ ++ +
+++ ++ +
++ ++ +/−
+++ +++ ++
+ ++ +
+++ +++ ++
++ +++ ++
Note: −, no staining; +/−, very sparse staining; +, faint staining; ++, moderate staining; +++, strong staining.
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Fig. 8. Immunolocalization of MMP-2 and MMP-9 in ovaries from neonatal guinea pigs in D-5 and P-5 groups.
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Fig. 9. Immunolocalization of MMP-2 and MMP-9 in ovaries from neonatal guinea pigs in D-10 and P-10 groups.
Conflict of interest The authors declare no conflict of interest. Acknowledgements We express our gratitude to Dr. Reinhold J. Hutz in the Department of Biological Sciences, University of Wisconsin-Milwaukee, USA, for reading the original manuscripts and offering valuable suggestions. This work was supported by the National Natural Science Foundation of China (No. 31172206).
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Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Potential roles of matrix metalloproteinases and characteristics of ovarian development in neonatal guinea pigs Junrong Li a,b , Ting Shen b , Guoyun Wu a , Quanwei Wei a , Dagan Mao a , Fangxiong Shi a,∗ a b
Laboratory of Animal Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China College of Agriculture and Bio-engineering, Jinhua Polytechnic, Jinhua 321017, China
a r t i c l e
i n f o
Article history: Received 27 May 2015 Received in revised form 1 July 2015 Accepted 20 July 2015 Available online 21 July 2015 Keywords: eCG Neonatal guinea pigs StAR PCNA MMP-2 MMP-9
a b s t r a c t The present study was conducted to investigate expression of matrix metalloproteinases (MMPs) and early ovarian development in neonatal guinea pigs. Thirty neonatal guinea pigs at 3 or 8 days of age were administrated 5 IU equine chorionic gonadotropin (eCG) or saline, and the ovaries were collected after 2 days of eCG. Serum concentrations of estradiol and progesterone were determined, and ovarian localization of StAR, MMP-2 and MMP-9 were analyzed by immunohistochemical staining. Results indicate that injection of eCG sensitized the neonatal ovary and elevated serum concentrations of estradiol and progesterone, but not enough to stimulate ovarian follicular development in the ovaries. MMP-2 and MMP-9 were both immunolocalized to the surface of granulosa cells of primary and secondary follicles, and high MMP-2 expression was accompanied by low StAR expression in eCG-treated ovaries. Collectively, we hypothesize that MMP-2, -9 and StAR are both involved in follicular atresia through their participation in cell proliferation and tissue remodeling. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Guinea pigs share some similarities in their reproductive cycle with humans, have a small ovulatory quota, and constitute a reliable animal model for studying human reproduction (Kulduk et al., 2014). However, ovarian development after birth in guinea pigs has been studied rarely, when compared with other rodents. Gonadotropins are basically used to induce multiple ovulations in animals, and equine chorionic gonadotropin (eCG) is a standard gonadotropin used to recruit additional follicles (Bó and Mapletoft, 2014). eCG plays a role similar to that of follicle-stimulating hormone (FSH) in superovulation (Mapletoft et al., 1990), but has a half-life up to 40 h (Zanetti et al., 2014; Murphy and Martinuk, 1991). eCG was effective in rats and mice, but eCG did not stimulate superovulation in adult guinea pigs (Suzuki et al., 2003). Our previous study showed that eCG played a role in adult guinea pigs similar to that of luteinizing hormone (LH); i.e., eCG induced luteinized unruptured follicle (LUF) syndrome in cyclic guinea pigs (Supplement Material). Our understanding of the sensitivity of
∗ Corresponding author. E-mail address: [email protected] (F. Shi). http://dx.doi.org/10.1016/j.tice.2015.07.005 0040-8166/© 2015 Elsevier Ltd. All rights reserved.
neonatal guinea pigs to eCG is lacking and the etiology of LUFs is still unknown. Matrix metalloproteinases (MMPs), which participate in tissue remodeling (Kessenbrock et al., 2010), are also involved in morphogenesis, apoptosis, ovulation and angiogenesis (Jones, 2014; Roy et al., 2006; Vu and Werb, 2000). MMPs play a critical role in follicular development in ovaries by remodeling the extracellular matrix (ECM) in the periovulatory period (Bagavandoss, 1998; Baker et al., 2000; Cooke et al., 1999; McCaffery et al., 2000). MMP-2 and MMP-9, both with a fibronectin-like domain in the middle of the catalytic domain (Bode et al., 1999), can degrade collagens, and possess laminin and aggrecan core proteins (Nagase et al., 2006), and MMP-2 and MMP-9 were both immunolocalized in developing follicular theca and stroma in rodents (Garcia et al., 1997). The intrafollicular levels of MMP-2 and MMP-9 both increased during the progression from normal to atresia ovarian follicles in sheep (Huet et al., 1997). A higher production of MMP-2 and MMP-9 was also accompanied by a higher follicular apoptosis rate in patients (Ben-Shlomo et al., 2003; Shalev et al., 2001). The objective of the present study was to investigate the role of eCG in neonatal guinea pigs, with a focus on expression of MMP-2 and MMP-9 in ovaries. In addition, immunolocalization of steroidogenic acute regulatory protein (StAR) and
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proliferating cell nuclear antigen (PCNA) were performed, since they are the indicators of steroidogenesis (Clark et al., 1997) and cell proliferation (Sun et al., 2014; Wildemann et al., 2003), respectively.
2. Materials and methods Thirty neonatal female Harley-White guinea pigs (Cavia porcellus) at 3 days of age were obtained from the Laboratory Animal Research Center of Zhejiang Chinese Medical University (Hangzhou, China). The guinea pigs were randomly divided into 5 groups (each n = 6): groups D-3, D-5, P-5, D-10 and P-10. The animals in group D-3 were sacrificed at 3 days of age; the animals in groups P-5 and D-5 were administered 5 IU eCG or saline at 3 days of age, and then sacrificed at 5 days; the animals in groups P-10 and D-10 were administered 5 IU eCG or saline at 8 days of age, respectively, and then sacrificed at 10 days of age. Blood samples were collected before scarification; and ovarian samples were collected immediately after scarification. The serum was separated from blood by centrifugation at 5000 g for 10 min, and the serum concentrations of estradiol and progesterone were measured (Adicon Clinical Laboratories INC, Hangzhou, China). The ovaries were fixed in 4% paraformaldehyde at room temperature for 36 h, and then kept in 70% alcohol for histologic and immunohistochemical (IHC) analyses. After fixation, ovarian samples were embedded in paraffin, sectioned serially at 5 m and stained with H&E. Sections were analyzed for observations of morphologic changes in ovaries after eCG injection. The follicular stages in neonatal guinea pigs were determined according to a previous study on mouse ovaries (Flaws et al., 1997). The numbers of primordial and antral follicles were both counted: primordial follicles in guinea pigs contained only one layer of granulosa cells, while antral follicles contained an antrum. In order to immunolocalized steroidogenesis in ovarian follicles, we performed IHC staining using monoclonal antibodies against StAR and PCNA (Santa Cruz Biotechnology Inc., TX, USA; SC25806, lot: I2308; SC7907, lot: L5346) with a strept avidin-biotin complex (SABC) kit (Boshide Biotechnology Inc., Wuhan, China; SA2002, lot: 10C09A). Ovarian expressions of MMP-2 and MMP9 were characterized by IHC staining with antibodies to MMP-2 and MMP-9 with an SABC kit (all from Fuzhou Maixin Biotech Co., Ltd., Fuzhou, China; lot: MAB-0244; lot: MAB-0245; lot: KIT5910). The antibodies were diluted to 1:200 in phosphate-buffer saline (PBS) containing 1% bovine serum albumin (BSA). Heatinduced epitope retrieval (HIER) was performed by heating the slides immersed in the 10 mM sodium citrate buffer (pH 6.0) at 100 ◦ C in a microwave oven for 8 min. The sections were mounted on slides coated with 3-aminopropyl-triethoxysilane (APES), and dried at 37 ◦ C for 24 h. The sections were incubated with the primary antibodies overnight at 4 ◦ C. The immunoreactivity was visualized using diaminobenzidine (DAB) (Sigma-Aldrich Corp., MO, USA) as substrate and counter-stained with hematoxylin. Normal rabbit serum, instead of primary antibody, was used as negative control. The stained ovarian samples were observed in a BX51 electrical microscope (Olympus Corporation, Tokyo, Japan). Images were recorded by an XC10 optical imaging system (Olympus Corporation), and analyzed on OlyVIA (version 2.4, Olympus Soft Imaging Solutions GmbH, Japan). Statistical analyses were performed on IBM SPSS Statistics 21 (Chicago, IL, USA). Differences were evaluated by one-way analysis of variance (ANOVA) with followed by Tukey’s test. P < 0.05 was considered to be significant.
Fig. 1. Serum concentrations of estradiol and progesterone in neonatal guinea pigs after eCG injection. D-5, 5-day-old guinea pigs with saline injection at 3 days of age; P-5: 5-day-old guinea pigs after injection with 5 IU eCG at 3 days of age; D10, 10-day-old guinea pigs with saline injection at 8 days of age; P-10: 10-day-old guinea pigs with injection of 5 IU eCG at 8 days of age. Each value is expressed as mean ± SEM. Different superscripts represent significant differences among categories (P < 0.05) as analyzed by Tukey test; while identical letters denote non significance (P > 0.05). F-test for homogeneity of variance, P > 0.05; W-test for normality of distribution, P > 0.05.
3. Results 3.1. Serum concentrations of estradiol and progesterone in neonatal guinea pigs As shown in Fig. 1, the serum concentrations of estradiol are significantly higher in group P-5 vs. group D-5, in group P-10 vs. group D-10, and in group P-10 vs. group P-5 (all P < 0.05); the serum concentrations of progesterone are significantly higher in group P5 vs. group D-5, in group P-10 vs. group D-10, in group P-10 vs. group P-5, and in group D-10 vs. group D-5 (all P < 0.05). 3.2. Observation of ovarian morphology in neonatal guinea pigs Few antral follicles were observed in the ovaries of group D3 (Fig. 2A). Specifically, primordial follicles were distributed close to ovarian walls (Fig. 2A1) and surrounded primordial follicles.
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Fig. 2. Changes in ovarian morphology in neonatal guinea pigs in D-3, D-5 and D-10 groups using H&E stain. D-3, normal 3-day-old guinea pigs. Oo: oocyte. GC: granulosa cell. TC: theca cell.
Moreover, the oocytes in primary follicles were closely located with granulosa cells (Fig. 2A2), while the secondary follicles were quite small in size (Fig. 2A3). Compared with group D-3, the number of antral follicles in group D-5 was significantly larger (Fig. 2B), the layer of granulosa cells and theca cells showed apparent boundaries (Fig. 2B1), and the secondary follicles were at different developmental stages (Fig. 2B2 and B3). Compared with group D-5, the number of antral follicles in group D-10 was significantly larger (Fig. 2C), the follicular antrum in group D-10 was larger in size (Fig. 2C1 and C2), and antral atretic follicles were observed in group D-10. Moreover, the granulosa cells were separated from
the layer of theca cells, and distributed in the follicular antrum (Fig. 2C3). 3.3. Changes in ovarian morphology in guinea pigs by eCG injection Compared with the ovaries in group D-5 (Fig. 3A), a significantly smaller number of antral follicles was observed in group P-5 (Figs. 3D and 4). The preantral follicles were contiguously arranged in ovaries in group P-5 (Fig. 3E and E1), unlike as in group D-5 where antral follicles were at a similar stage (Fig. 3B
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Fig. 3. Changes in ovarian morphology in neonatal guinea pigs in D-5 and P-5 groups using H&E stain. Oo: oocyte; GC: granulosa cell; TC: theca cell; AF: antral follicle; PmF: primordial follicle.
and B1). Non-antral atretic follicles were observed in both groups D-5 and P-5 (Fig. 3A1 and D1), but the number of primordial follicles in group P-5 was significantly smaller than in group D-5 (Fig. 4). Compared with the ovaries in group D-10 (Fig. 4A), the number of antral follicles in group P-10 was significantly smaller (Fig. 5A). The antral secondary follicles in the ovaries of group D-10 were at different growth stages (Fig. 4B), as indicated by the fact that the layer of granulosa cells was thinner and closely connected to the layer of theca cells (Fig. 4B1). However, the antral follicles in group P-10 were smaller in size vs. in group D-10 (Fig. 4E), and were mostly irregularly-shaped (Fig. 4E1). Compared wtith group D-10, the number of primordial follicles in group P-10 was significantly larger (Fig. 5B) and located in the middle of the ovaries (Fig. 4F1).
Antral atretic follicles were observed in group D-10 (Fig. 4A1), but not in group P-10 (Fig. 4D1). 3.4. Immunolocalization of StAR and PCNA in ovaries from guinea pigs after eCG injection StAR and PCNA were mainly expressed in granulosa cells in small antral secondary follicles of ovaries in group D-3 (Fig. 6A and B). StAR and PCNA were both expressed in antral and nonantral follicles in group D-5, and mainly immunolocalized in layers of granulosa cells in the ovaries (Fig. 6D and E). PCNA was immunolocalized in layers of granulosa cells also, but StAR was mainly expressed in layers of theca cells in primary follicles of ovaries in group P-5 (Fig. 6G and H).
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Fig. 4. Changes in ovarian morphology in neonatal guinea pigs in D-10 and P-10 groups using H&E staining. Oo: oocyte; GC: granulosa cell; TC: theca cell; AF: antral follicle; PmF: primordial follicle.
StAR and PCNA were both expressed in ovaries in group D10, but StAR was barely expressed in granulosa cells separated from theca cells in atretic follicles (Fig. 6J and K). StAR and PCNA were both mildly expressed in ovaries in group P-10, and primarily expressed in granulosa cells of small secondary follicles (Fig. 6M and N). 3.5. Expression of MMP-2 and MMP-9 in ovaries from guinea pigs after eCG injection MMP-2 and MMP-9 were both expressed in ovaries in group D3 (Fig. 7A and B) and D-5 (Fig. 8A and B), and mainly expressed in the layer of granulosa cells in primary and secondary follicles (Figs. 7A1, B1 and 8A1, B1). MMP-2 and MMP-9 were both expressed in ovaries in group P-5 (Fig. 8D and E), and MMP-2 was
also immunolocalized in theca cells of secondary follicles (Fig. 8D1). MMP-2 was expressed in both granulosa cells and theca cells of antral follicles in group P-5 (Table 1), but the expression level of MMP-9 in group P-5 was similar (Table 2), group compared with D-5. MMP-2 and MMP-9 were both immunolocalized in primary and secondary follicles in ovaries of group D-10 (Fig. 9A and B), and mainly expressed in granulosa cells (Fig. 9A1 and B1). MMP-2 and MMP-9 were also expressed in primary and secondary follicles in ovaries of group P-10 (Fig. 9D and E), and both were observed in the layers of granulosa cells (Fig. 9D1 and E1). The MMP-2 and MMP-9 expressions in antral follicles were higher (Table 1), but the MMP-9 expression in primary follicles was lower in group P-10 (Table 2), compared with group D-10.
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Fig. 5. Changes in the numbers of antral and primordial follicles in neonatal guinea pig ovaries after eCG injection. Each value is expressed as mean ± SEM. Different superscripts represent significant differences among categories (P < 0.05) analyzed with the Tukey test; while identical letters denote non significance (P > 0.05). F-test for homogeneity of variance, P > 0.05; W-test for normality of distribution, P > 0.05.
4. Discussion Neonatal guinea pigs at 3 and 8 days of age were sensitive to eCG, as the serum concentrations of progesterone and estradiol were elevated 2 days following injection of 5 IU eCG. The serum progesterone concentration in eCG-treated guinea pigs at 10 days of age was the highest. eCG might play a function similar to LH in
neonatal guinea pigs, which we observed in cyclic adult guinea pigs in our previous study (Li et al., 2015). The antral secondary follicles already appeared in ovaries of 3-day-old guinea pigs, and the follicular development might be initiated in the prenatal period. The ovarian morphology obviously changed during growth of neonatal guinea pigs. Specifically, the number of antral follicles increased steadily at 3, 5
Table 1 Intensity of IHC staining of MMP-2 in ovaries from neonatal guinea pigs after eCG treatment. Cell types
Primordial follicles Oocytes Primary follicles Oocytes GC TC Antral follicles Oocytes GC TC
Control groups
eCG groups
D-3
D-5
D-10
P-5
P-10
+++
+++
+++
++
+++
+++ ++ +/−
+++ ++ +
+++ ++ +
+++ ++ ++
++ ++ ++
+++ +++ +
+++ ++ ++
++ ++ +
++ +++ +++
+++ +++ +++
Note: −, no staining; +/−, very sparse staining; +, faint staining; ++, moderate staining; +++, strong staining.
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Fig. 6. Immunolocalization of StAR and PCNA from ovaries of neonatal guinea pigs. NC: negative control.
and 10 days age; while the percentage of primordial follicles was reduced. In contrast to normal ovaries in 5-day-old guinea pigs, antral follicles were not observed in the ovaries treated at 3 days of age, while the number of primary follicles increased. The results indicated that eCG might inhibit the development from primary to secondary follicles, but promote the recruitment of primary follicles in neonatal ovaries. No large antral secondary follicles were observed in ovaries after eCG injection at day 8. The number of antral follicles was
also lower than in normal 10-day-old ovaries. The number of primary follicles greatly increased in eCG-treated ovaries and the primary follicles were evenly distributed in the middle portions of the ovaries. This indicated that eCG did not stimulate follicular development like FSH, since the antral follicles existing in eCGtreated ovaries might appear even before administration. StAR and PCNA were both expressed in ovaries of 3-day-old guinea pigs, while cellular proliferation of granulosa cells in primary follicles was initiated and accompanied development of
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Fig. 7. Immunolocalization of MMP-2 and MMP-9 in ovaries from neonatal guinea pigs in D-3 group.
primary follicles in neonatal ovaries (Clark et al., 1997). This process might require steroidogenic support (Wildemann et al., 2003). StAR was scarcely expressed in preantral primary follicles of 5day-old ovaries after stimulation with 5 IU eCG. eCG might inhibit steroidogenesis in granulosa cells, and then the development from preantral to antral follicles, which is a potential reason for the absence of antral follicles in treated ovaries. StAR and PCNA were both expressed in atretic follicles in normal 10-day-old ovaries, which indicated that StAR and PCNA were both involved in the atretic process (Xu et al., 2011; Wang et al., 2010). StAR was moderately expressed in ovaries after eCG injection at day 8, which might be a reason for the absence of large antral follicles in eCG-treated ovaries. Thus, eCG might inhibit follicular development and even the activity of FSH in ovaries. MMP-2 and MMP-9 were both expressed in primary and secondary follicles in normal ovaries of 5- or 10-day-old guinea pigs. This result indicated that both MMP-2 and MMP-9 were involved in follicular development in ovaries through their participation in cell proliferation and tissue remodeling (Sternlicht and Werb,
2001; Huet et al., 1997). Both MMPs were immunolocalized on the surface of granulosa cells, and might be involved in regulation of extracellular granulosa cells (Sternlicht et al., 2000). MMP-2 was strongly expressed in eCG-treated ovaries at 3 days of age, and might be highly sensitive to eCG or played a prominent role in tissue remodeling in eCG-treated ovaries. Meanwhile, the high MMP-2 expression was accompanied by the low StAR expression in eCG-treated ovaries, which indicated that the tissue remodeling in ovaries might not depend on steroidogenesis. Compared with normal 10-day-old ovaries, the expression of MMP-9 in primary follicles was reduced in eCG-treated ovaries at 8 days of age, which may be a reason for the small number of antral follicles, and indicated that MMP-2 might be more involved in tissue remodeling in treated ovaries. In conclusion, neonatal ovaries in guinea pigs are already sensitive to the eCG, but follicular development in ovaries was not stimulated by eCG. MMP-2, -9 and StAR might be jointly involved in follicular atresia through their participation in cell proliferation and tissue remodeling.
Table 2 Intensity of IHC staining in MMP-9 in ovaries from neonatal guinea pigs after eCG treatment. Cell types
Primordial follicles Oocytes Primary follicles Oocytes GC TC Antral follicles Oocytes GC TC
Control groups
eCG groups
D-3
D-5
D-10
P-5
P-10
++
+++
+++
+++
+++
++ ++ +/−
+++ + +/−
+ +++ ++
+++ ++ +
+++ ++ +
++ ++ +/−
+++ +++ ++
+ ++ +
+++ +++ ++
++ +++ ++
Note: −, no staining; +/−, very sparse staining; +, faint staining; ++, moderate staining; +++, strong staining.
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Fig. 8. Immunolocalization of MMP-2 and MMP-9 in ovaries from neonatal guinea pigs in D-5 and P-5 groups.
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Fig. 9. Immunolocalization of MMP-2 and MMP-9 in ovaries from neonatal guinea pigs in D-10 and P-10 groups.
Conflict of interest The authors declare no conflict of interest. Acknowledgements We express our gratitude to Dr. Reinhold J. Hutz in the Department of Biological Sciences, University of Wisconsin-Milwaukee, USA, for reading the original manuscripts and offering valuable suggestions. This work was supported by the National Natural Science Foundation of China (No. 31172206).
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