Background
In mammals, the primordial follicle pool present at birth represents the total germ cell population available to a female during her entire reproductive life. The differentiation of primordial germ cells into functional oocytes contained in primordial follicles is poorly understood. In the mouse embryo, primordial germ cells migrate to the female genital ridge and are then referred to oogonia once they colonize the ovary [
18]. Oogonia develop in connected clusters known as cysts until 13.5 days post coitum (dpc) in the mouse and then become oocytes as they begin to enter meiosis [
19]. Meiotic entry occurs in a wave from the anterior to the posterior side of the ovary [
2,
15]. The oocytes proceed through prophase I of meiosis progressing through a series of sub-phases starting with pre-meiotic interphase and then moving through leptotene, zygotene, pachytene, and eventually arresting at the diplotene stage [
1]. The extended diplotene arrest often lasting years (until ovulation) is sometimes referred to as dictyate [
5]. The germ cells enter diplotene arrest beginning at 17.5 dpc, which corresponds with the start of cyst breakdown [
1,
8,
21]. As more germ cells arrive at diplotene and cysts begin to break down to form primordial follicles, some oocytes experience programmed cell death [
20]. In late fetal and early neonatal development, cysts break apart into individual oocytes and are surrounded by somatic pre-granulosa cells [
13,
20]. This results in primordial follicles each consisting of a diplotene arrested oocyte enclosed by several granulosa cells.
Mutations that disrupt meiotic prophase I progression in females affect fertility. For example, in females mutant for genes involved in recombination and repair, the germ cells do not progress beyond the pachytene stage of prophase I and eventually die resulting in infertility [
18]. Also, inhibition of Synaptonemal Complex Protein 1 mRNA (
Sycp1), found in synaptonemal complexes which are protein structures that hold the homologous chromosome pairs together at the pachytene stage, caused premature arrival at the diplotene stage and premature primordial follicle formation suggesting a link between cell cycle stage and primordial follicle formation [
17]. However, in
Stra8 mutants meiotic entry is blocked but primordial follicles still form implying that meiosis and follicle formation are independent [
7]. We found a small subset of primordial follicles with oocytes at prediplotene stages supporting the idea that oocytes do not have to reach the diplotene stage before follicles form [
8].
Previous work from our lab demonstrated that estrogen or progesterone can reduce cyst breakdown and primordial follicle formation and together have an additive effect [
3]. There is also some evidence that steroid hormones can affect progression through meiotic prophase I. For example, in cows, high levels of estradiol (E
2) and progesterone (P
4) were associated with a delay in reaching the diplotene stage [
26]. Supporting this, treatment of mouse embryos with the estrogenic compound, bisphenol A (BPA) caused defects in meiosis suggesting that E
2 signaling could be involved in regulating meiotic progression [
23]. Estrogen receptor 2 (
Esr2) mutants had meiotic defects similar to BPA treated animals suggesting that BPA acts as an ESR2 antagonist. Work from our lab found that in fetal organ culture progesterone but not estradiol delayed transit though meiotic prophase I [
8].
One of the most common techniques used for meiotic staging is the histological method of hematoxylin and eosin (H&E) staining that requires embedding in paraffin, sectioning, staining and then analysis of ovary sections for meiotic stage. This method is tedious, time consuming and results in a loss of three dimensional structural information. A more recently used molecular technique is the surface spread assay involving lysis of the ovary to open the cells allowing labeling of chromosomes with a fluorescent marker. While this method greatly improves the ability to identify oocytes in stages of meiotic prophase I, the ovary is completely disaggregated resulting in the loss of any structural information. One reported molecular diplotene arrest marker is MSY2, an RNA binding protein expressed in germ cells [
9]. MSY2 is believed to be involved in regulating mRNA stability in growing oocytes and when the gene is deleted, females become sterile [
24]. Several groups have used MSY2 protein expression as an indicator that an oocyte has reached the diplotene stage of meiotic prophase I [
17,
22]. Another protein used as a marker for meiotic prophase I staging is SYCP3. As the oocytes reach pachytene, the synaptonemal complex forms holding homologous chromosomes together and SYCP3 localizes in between the chromosomes. At diplotene, homologous chromosomes begin to separate remaining attached only at points of crossing over and SYCP3 becomes diffuse except for a few sites of strong staining [
4,
6].
The objective of the work presented here was to test effects of steroid hormones, estrogen and progesterone on meiotic prophase I progression using available molecular tools.
Materials and methods
Animals
CD-1 mice used for RNA studies were obtained from Charles River Laboratories and C57BL/6 mice used for all other studies were obtained from Jackson Laboratories. Mice were housed and bred at a controlled photoperiod (14 h light, 10 h dark), temperature (21–22 °C), and humidity with food and water available ad libitum. Females were mated with males of the same strain and checked daily for vaginal plugs. Noon on the day of vaginal plug detection was designated as 0.5 dpc. Birth usually occurred at 19.5 dpc and was designated as postnatal day (PND) 1. Pregnant mice were euthanized by CO2 asphyxiation for fetal ovary collection. For neonatal ovary collection, pups were euthanized by decapitation on the appropriate day. All animal protocols were approved by the Syracuse University Institutional Animal Care and Use Committee.
Study design
Mouse ovary organ culture was used to investigate the effects of hormones on oocyte meiotic prophase I progression. Ovaries were harvested at 17.5 dpc and cultured for 5 days in DMSO, E2, P4 or both hormones at 10− 6 M. Ovaries were collected and labeled with antibodies against MSY2 and TRA98 or SYCP3 and VASA using immunocytochemistry. Msy2 mRNA expression during fetal and neonatal oocyte development was examined using RT-PCR. The expression of MSY2 and SYCP3 protein was followed during oocyte development using whole mount immunocytochemistry. SYCP3 protein was also followed over time using the meiotic surface spread technique.
RNA isolation
Fetal (13.5 dpc-18.5 dpc) and neonatal (PND1-PND5) ovaries were dissected in PBS, placed in RNAlater, flash frozen in liquid nitrogen and stored at − 80 °C. Total RNA was isolated using Qiagen’s RNeasy Mini kit following the manufacturers instructions (n = 3; 50 or 100 ovaries per pool for neonatal and fetal ovaries respectively). Ovaries were briefly lysed and homogenized using a motor pestle on ice and the mixture was then applied to a QIAshredder column. The ovarian tissue sample in the QIAshredder column was then centrifuged at 11,000 g for 2 min. To isolate the RNA, the resulting flow-through was applied to an RNeasy mini column which allowed the RNA to bind to the filter cartridge. RNA was eluted by washing from the filter and was concentrated using an RNeasy MinElute kit. The RNA which was isolated was briefly applied to an RNeasy MinElute spin column and after washing, RNA was eluted using 14 μl of RNase-free water. The RNA concentration in the elutant was determined using an ND-1000 Spectrophotometer (λ = 260/280 nm; Nanodrop Technologies, Inc., Wilmington, DE).
First strand cDNA synthesis and real-time polymerase chain reaction (PCR)
Total RNA (0.5 μg) was reverse transcribed into cDNA using the Superscript III One-Step RT-PCR System. The cDNA was diluted in RNase-free water (1:25). 2 μl of diluted cDNA was amplified on a Rotor-Gene 3000 using Quantitect™ SYBR Green PCR kit and custom designed primers for Msy2 (forward primer: 5′ CCC TGG CAA CCA GGC GAC GG 3′; reverse primer: 5′ TGA CTG TGC CCA GGA CTT GGA TTG 3′; NCBI Genbank accession number NM_016875), and β-actin (forward primer: 5′ AGT GTG ACG TTG ACA TCC GTA 3′; reverse primer: 5′ GCC AGA GCA GTA ATC TAA TTA T 3′; NCBI Genbank accession number NM_007393). The cycling program consisted of a 15 min hold at 95 °C and 45 cycles of: denaturing at 95 °C for 15 s, annealing at 58 °C for 15 s, and extension at 72 °C for 20 s at which point data were acquired. Determination of product melt conditions was done using a temperature gradient from 72 °C to 99 °C with a 1 °C increase at each step. β-actin expression remained constant across all ages and therefore each sample was normalized to β-actin before quantification.
Immunocytochemistry
Once ovaries were harvested, they were fixed with 5.3% EM grade formaldehyde in PBS overnight at 4 °C and immunostained as previously described [
16]. Briefly, ovaries went through a series of washes at room temperature in 0.1% Triton X-100 in 1X PBS (PT) and then PT + 5% bovine serum albumin (BSA). Following washes, ovaries were incubated overnight with primary antibodies diluted in PT + 5% BSA at 4 °C (see Table
1 for antibodies and dilutions). After overnight incubation in primary antibodies, ovaries were washed in PT + 1% BSA treated with RNase A and labeled with propidium iodide or TOTO3. Ovaries were then incubated with pre-absorbed secondary antibodies (see Table
2 for secondary antibodies) at a dilution of 1:200 overnight at 4 °C. Negative controls using only secondary antibodies were previously tested in the lab for all antibodies used. Ovaries were washed in PT + 1% BSA, rinsed in PBS, placed in Vectashield, mounted and observed by confocal microscopy on a Zeiss LSM 710 confocal microscope.
Table 1Primary Antibodies and Dilutions Used
MSY2 (C-15) | Santa Cruz Biotechnology | sc-21,316 Lot #: C0615 | Goat | 1:100 |
SYCP3 (D-1) | Santa Cruz Biotechnology | sc-74,569 Lot #: J1314 | Mouse | 1:200 (whole mount) 1:50 (surface spreads) |
TRA98 | B-Bridge | 73–003 Lot #: 141113A | Rat | 1:500 |
VASA | Abcam | ab-13,840 Lot #: GR172688–1 | Rabbit | 1:250 |
Table 2Secondary Antibodies and Dilutions Used
Alexa Fluor 488 Donkey Anti-Goat | Molecular Probes/Invitrogen | A11055 | Donkey | 1:200 |
Alexa Fluor 488 Donkey Anti-Rat | Molecular Probes/Invitrogen | A21208 | Donkey | 1:200 |
Alexa Fluor 488 Goat Anti-Mouse | Molecular Probes/Invitrogen | A11029 | Goat | 1:200 |
Alexa Fluor 488 Goat Anti-Rabbit | Molecular Probes/Invitrogen | A11008 | Goat | 1:200 |
Alexa Fluor 568 Donkey Anti-Goat | Molecular Probes/Invitrogen | A11057 | Donkey | 1:200 |
Alexa Fluor 568 Goat Anti-Mouse | Molecular Probes/Invitrogen | A11004 | Goat | 1:200 |
Meiotic surface spreads
Ovaries were harvested, incubated in Hypotonic Extraction Buffer (30 mM Tris, 50 mM sucrose, 17 mM trisodium citrate dihydrate, 5 mM EDTA, 0.5 mM DTT, and 0.5 mM phenylmethylsulphonyl fluoride (PMSF), pH 8.2) and then teased apart in 100 mM sucrose. The cell suspension was dried down and fixed in 1% paraformaldehyde. Slides were incubated overnight in a humidity chamber at 37 °C. Slides were then air dried, washed in 0.4% PhotoFlo, air dried again and stored at − 20 °C until staining. Slides were washed in PBS, blocked with 2.5% goat serum and stained with antibodies against SYCP3 (see Table
1) diluted in 2.5% goat serum in a humidity chamber overnight at 4 °C. Subsequently, slides were washed with 0.1% Tween in PBS and then incubated Alexa Fluor 488 Goat Anti-Mouse secondary antibodies (see Table
2) diluted in 2.5% goat serum for 1 hour. Slides were washed with 0.1% Tween in PBS, mounted in a 1:1 solution of Vectashield and 2 μg/ml DAPI and stored at − 20 °C.
In vitro ovary organ culture
Ovaries dissected at 17.5 dpc were placed in culture. Ovaries were cultured in 4-well culture plates in drops of media on 0.4 μM floating filters (Millicell- CM; Millipore Corp., Bedford, MA) in 0.4 ml DMEM-Ham’s F-12 media supplemented with penicillin-streptomycin, 5X ITS-X (Life Technologies, Inc., Grand Island, NY), 0.1% BSA, 0.1% albumax, and 0.05 mg/ml L-ascorbic acid. E2 and P4 (Sigma Chemical Co., St. Louis, MO) were dissolved in dimethylsulfoxide (DMSO) at a concentration of 0.1 M and then added to culture media to achieve the desired final concentration. DMSO was added to media at the same percentage as a vehicle control. Ovaries were exposed daily to DMSO, E2, P4 or both hormones at 10− 6 M (n = 5 ovaries per treatment group). Ovaries were divided randomly among the treatment groups. Ovaries were fixed in formaldehyde and immunostained as described above.
Statistical analysis
Data are represented as mean ± SEM of nontransformed data. Statistical analyses using transformed data were performed using GraphPad Prism version 6 (GraphPad Software, San Diego, CA). Statistical differences (P < 0.05) among the means were evaluated using one-way ANOVA followed by Newman-Keuls multiple comparisons test. Effects of E2 and P4 on MSY2 and SYCP3 expression were analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test. Statistical analyses of real time PCR data were performed using Statview 5.0.1 (SAS Institute Inc., Cary, NC). Differences between fold increases in mRNA levels over various time points were evaluated by one-way ANOVA followed by Bonferroni-Dunn’s post hoc test (P < 0.0005).
Discussion
Here, we show that MSY2 expression in oocytes is reduced by progesterone but not estradiol alone or estradiol and progesterone in combination using ovary organ culture. In addition, while MSY2 upregulation correlates with arrival at the diplotene stage, is a not a diplotene stage marker as some pre-diplotene oocytes highly express MSY2. SYCP3 can be used to follow progress of oocytes through meiotic prophase I using both surface spreads and whole mount immunostaining with the pachytene stage especially prominent. Finally, estradiol and progesterone together but not either hormone individually delay prophase I progression as determined by the expression pattern of SYCP3 in whole mount immunostaining.
Our data suggest that MSY2 protein levels increase as oocytes approach the diplotene phase of meiotic prophase I but is not a diplotene stage marker per se. As shown in Fig.
2b, MSY2 is strongly expressed in oocytes even at 15.5 dpc before any oocytes have reached the diplotene stage. MSY2 is a conserved RNA binding protein specifically expressed in germ cells and required for fertility [
9,
24]. In male germ cells it is important post-meiotically during spermiogenesis [
25]. In oocytes, MSY2 protein regulates mRNA stability as oocyte increases in size [
14].
Msy2 mutant oocytes have many abnormalities such as aberrant spindle formation and chromosome congression during meiosis II, however, there is no evidence for a role of MSY2 in meiotic prophase I. Likely, the expression of MSY2 is upregulated during meiotic prophase I in preparation for future oocyte growth and later meiotic functions.
Previous research has shown that estrogen and progesterone have negative effects on the development process of female germ cells. Progesterone and estradiol, the phytoestrogen genistein, as well as synthetic estrogens all disrupt cyst breakdown and follicle formation [
3,
11,
12] thereby leading to a potential decrease in viable egg cells later in life. In some instances, the number of oocytes present is also affected, but not as consistently. Here, ovaries were treated with estradiol alone, progesterone alone, or both estradiol and progesterone and effects on meiotic progression examined using SYCP3 expression in whole mount immunostaining. The number of oocytes found in the pachytene stage significantly increased only in the estradiol and progesterone treatment group indicating that treatment with both hormones delayed meiotic progression. Previous research has shown that when pregnant female mice were exposed to Bisphenol A (BPA), an estrogenic chemical, meiotic progression was disrupted by way of a disturbance of the synapsis and recombination of chromosome homologs [
23]. Another study showed that progesterone acts through the progesterone receptor membrane component 1 (PGRMC1) to significantly delay or completely disrupt meiotic progression and therefore disrupt primordial follicle assembly [
10]. The results of our organ culture agreed with these findings.
Our previous work investigating prophase I progression using standard histology demonstrated that progesterone but not estradiol or the combination of estradiol and progesterone delayed meiotic progression [
8]. However, in the work presented here only the combination of estradiol and progesterone significantly affected progression through meiosis. One difference between the two studies is the mouse strain used. The CD1 outbred strain was used in our earlier study while here we used the B6 inbred strain. Interestingly, we also found that only progesterone alone reduced MSY2 expression.
We used two different molecular techniques to follow prophase I progression during perinatal oocyte development. The surface spread technique combined with a synaptonemal complex marker such as SYCP3 allows precise identification of prophase I substages. However, all structural information is lost with this method. The second technique, also using SYCP3 is whole mount immunostaining which preserves three dimensional structural information but does not allow precise identification of prophase substages though the pachytene stage is easy to identify. Using both techniques we found the largest percentage of pachytene oocytes at PND1 (~ 45% in surface spreads and ~ 55% in immunostaining). Thus, both techniques were able to be used to provide information regarding meiotic progression.
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