- Email: [email protected]
Immunotherapy using inhibin antiserum enhanced the efficacy of equine chorionic gonadotropin on superovulation in major inbred and outbred mice strains
Accepted Manuscript Immunotherapy using inhibin antiserum enhanced the efficacy of equine chorionic gonadotropin on superovulation in major inbred and outbred mice strains Toru Takeo, Naomi Nakagata PII:
S0093-691X(16)30119-4
DOI:
10.1016/j.theriogenology.2016.04.076
Reference:
THE 13656
To appear in:
Theriogenology
Received Date: 3 February 2016 Revised Date:
25 April 2016
Accepted Date: 25 April 2016
Please cite this article as: Takeo T, Nakagata N, Immunotherapy using inhibin antiserum enhanced the efficacy of equine chorionic gonadotropin on superovulation in major inbred and outbred mice strains, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.04.076. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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New superovulation technique Inhibin antiserum + Equine chorionic gonadotropin
Inbred mice: A/J, BALB/cByJ, C3HeJ, DBA/2J, FVB/NJ outbred mouse: CD1
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No. of ovulated oocytes: 7-29 oocytes
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Conventional superovulation technique Equine chorionic gonadotropin
No. of ovulated oocytes: 24-90 oocytes
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Inhibin antiserum improves the superovulation technique. Coadministration of inhibin antiserum and gonadotropin produces many oocytes. The effect is effective to major inbred and outbred mouse strains. The oocytes normally fertilize and develop to offspring.
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Immunotherapy using inhibin antiserum enhanced the efficacy of
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equine chorionic gonadotropin on superovulation in major inbred and
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outbred mice strains
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Running title: Improvement of superovulation by inhibin antiserum
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Summary sentence: Coadministration of inhibin antiserum and equine
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chorionic gonadotropin improves superovulation technique and is very
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effective in obtaining high number of ovulated oocytes in major inbred
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and outbred mice strains.
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Key words: Superovulation, inhibin antiserum, equine chorionic
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gonadotropin, oocytes, mice, embryo production, mouse strain, mouse
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repository
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Toru Takeo and Naomi Nakagata
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Division of Reproductive Engineering, Center for Animal Resources and
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Development (CARD), Kumamoto University, Chuo-ku, Kumamoto,
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Japan
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Correspondence:
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Naomi Nakagata, E-mail: [email protected], Telephone:
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+81-96-373-6570, Fax: +81-373-6566
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ABSTRACT
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Improvement of the superovulation technique will help to enhance the
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efficiency of embryo and animal production. Blocking inhibin using
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inhibin antiserum (IAS) is known to promote follicular development by
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increasing the level of follicular stimulating hormone. Previously, we
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reported that coadministration of IAS and equine chorionic gonadotropin
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(eCG) produced more than 100 oocytes from a single female C57BL/6
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mouse at 4 weeks old. The oocytes derived from the IAS + eCG (IASe)
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treatment were able to fertilize and develop normally into offspring. In this
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study, we examined the effect of IASe treatment on the numbers of
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ovulated oocytes in major inbred (A/J, BALB/cByJ, C3HeJ, DBA/2J, and
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FVB/NJ) and outbred (CD1) mice strains at 4 weeks old. We confirmed
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the fertilization and developmental ability of the IASe-derived oocytes.
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IASe treatment ovulated 1.5 to 3.2 times higher numbers of oocytes than
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eCG treatment alone. The fertilization rate of IASe-derived oocytes was
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similar to that of eCG-derived oocytes. In vitro and in vivo developmental
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rates of the embryos derived from IASe was similar to the rates of
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embryos derived from eCG. We have shown that superovulation by IASe
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is very effective in obtaining high numbers of ovulated oocytes from small
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numbers of oocyte donor in a number of mice strains. The superovulation
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technique will contribute to the archiving of cryopreserved embryos of
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genetically engineered mice using small numbers of donors and has the
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potential to produce more live animals for rederivation of the archived
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mouse lines in mouse repositories.
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INTRODUCTION
Mouse repositories that support the production, preservation, and
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distribution of genetically engineered mice have been established to
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improve the accessibility of these mice to the scientific community [1].
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The archived lines of genetically engineered mice are published by the
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International Mouse Strain Resource (IMSR; http://www.findmice.org/).
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Researchers can order and obtain mouse lines via the IMSR.
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Reproductive technology is a powerful tool to efficiently manage
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genetically engineered mice in mouse repositories [2]. In vitro fertilization
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(IVF) using sperm and oocytes harvested from genetically engineered
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mice can readily produce a huge number of embryos. The embryos can be
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transferred to recipients to obtain the numbers of offspring required for a
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planned animal experiment or they can be preserved in liquid nitrogen for
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later use. The application of reproductive techniques for animal production
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allows the precise management of size and schedule of experiments using
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genetically engineered mice.
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The number of ovulated oocytes is a bottleneck that impacts the
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numbers of embryos and animals that can be produced by reproductive
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technology. It is important, therefore, to increase the number of ovulated
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oocytes using superovulation techniques, which artificially promote
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folliculogenesis
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Superovulation has been used successfully to obtain oocytes for the
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production of genetically engineered mice [3]. Traditionally, a regimen of
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equine chorionic gonadotropin (eCG) and human chorionic gonadotropin
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(hCG) has been used to induce superovulation in mice [4]; however, the
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response to this technique is known to vary among inbred and hybrid
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mouse strains [5, 6].
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Administration of inhibin antiserum (IAS) has been shown to induce
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superovulation in several species [7]. IAS neutralized inhibin, which is a
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non-steroidal hormone that acts on pituitary cells to suppress the
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production of follicle-stimulating hormone (FSH) [8]. The blocking of
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inhibin by IAS promoted follicular development by facilitating the
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production of FSH [9, 10]. Recently, we developed a novel superovulation
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technique by the coadministration of IAS and eCG [11], which yielded
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more than 100 oocytes from a single female C57BL/6 mouse at 4 weeks
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old. The ovulated oocytes were fertilized in vitro and live pups developed
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after IVF and embryo transfer [11]. However, the efficacy of the IAS +
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eCG (IASe) treatment on female mice of major inbred and outbred strains
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has not been examined so far.
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In this study, we investigated the effect of IASe treatment on the
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number of ovulated oocytes in major inbred and outbred mouse strains of
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A/J, BALB/c, C3H, CD1, DBA, and FVB mice. The ovulated oocytes
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were used for IVF, and the embryos were cultured and transferred to
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confirm their fertilization and developmental ability.
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MATERIALS AND METHODS
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Animals
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Inbred mouse strains of A/J (SLC, Shizuoka, Japan), BALB/cByJ
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(CLEA Japan, Tokyo, Japan), C3HeJ (CLEA Japan), DBA/2J (CLEA
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Japan), and FVB/NJ (CLEA Japan) and an outbred mouse strain of CD1
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(Charles River Japan, Yokohama, Japan) were purchased and used as
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donors of sperm (from male mice at 12–15 weeks old) and oocytes (from
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female mice at 4 weeks old). ICR mice (CLEA Japan) at 8–16 weeks old
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were used as recipients of 2-cell embryos. All the mice were housed in a
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specific-pathogen-free room under a 12-h dark-light cycle (light from
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07:00 to 19:00) at a 22 ± 1°C with ad libitum food and water. The Animal
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Care and Use Committee of the Kumamoto University School of Medicine
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approved the protocols for the experiments using animals. The mice were
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properly handled and euthanized by the person who was admitted and
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directed by the Animal Care and Use Committee of Kumamoto University.
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Medium
IAS was prepared as described previously and preserved at -20oC
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before use [9, 10]. Sperm were preincubated in a modified Krebs–Ringer
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bicarbonate solution (TYH) containing 1.0 mg/mL polyvinyl alcohol
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(Sigma) and 0.75 mM methyl-β-cyclodextrin (Sigma) as sperm
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preincubation medium [12, 13]. Calcium-enhanced human tubal fluid
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(mHTF) containing 0.25 mM reduced glutathione (Sigma) was used as
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fertilization medium [14-16]. Potassium simplex optimized medium
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(KSOM) was used to handle and culture the 2-cell embryos to the
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blastocyst stage [17]. All media were prepared and stored at 4oC no longer
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than 3 months before use. Sperm preincubation medium, fertilization
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medium and embryo culture medium were preincubated in CO2 incubator
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(5% CO2 and 95% air) at 37oC for 30 min before use.
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Superovulation
Superovulation was performed as described in our previous study [11].
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Briefly, immature female mice at 4 weeks old were administered 7.5 IU
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eCG (ASKA Pharmaceutical Co. Ltd, Japan) or IASe (mixed solution of
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0.1 mL IAS and 3.75 IU eCG) intraperitoneally. Forty-eight hours after the
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injection of eCG or IASe, the mice were administered 7.5 IU hCG (ASKA
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Pharmaceutical Co. Ltd, Japan) intraperitoneally. At 17 h after hCG
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administration, the mice were euthanized by cervical dislocation and the
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oviducts were collected quickly and transferred to a fertilization dish
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covered with paraffin oil. Under microscopic observation, cumulus–oocyte
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complexes were collected from the oviducts by dissecting needle and
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transferred to a 200-µL drop of fertilization medium. The numbers of
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ovulated oocytes and their ability to be fertilized were examined in each
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group.
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In vitro fertilization Male mice were euthanized by cervical dislocation and the cauda
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epididymides were collected and transferred to a dish of sperm
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preincubation medium covered with paraffin oil. Aliquots of sperm were
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collected from the cauda epididymides using a dissecting needle and
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transferred to a 100-µL drop of sperm preincubation medium. Sperm were
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preincubated for 60 min to induce capacitation and then added to the
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fertilization drop containing the cumulus–oocyte complexes and cultured
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with oocytes at 37°C in an atmosphere containing 5% CO2 for 3 h. The
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concentration of motile sperm was measured by computer-assisted sperm
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analysis (IVOS, Hamilton Thorn Inc.) before insemination and then
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adjusted to 400–800 motile sperm/µL in fertilization medium. At 3 h after
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insemination, oocytes were washed in three drops of mHTF (80 µL) and
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the number of ovulated oocytes was examined. At 24 h after insemination,
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fertilization rates were calculated as the total number of 2-cell embryos
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divided by the total number of inseminated oocytes multiplied by 100.
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Embryo culture and transfer To evaluate the development of the 2-cell embryos produced by IVF,
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we performed embryo culture and transfer. After IVF, the 2-cell embryos
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were divided into two groups; one that was cultured to the blastocyst stage
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in a 100-µL drop of KSOM and another that was transferred into the
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oviducts of ICR females (10 embryos/oviduct) on the day a vaginal plug
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was found (day 1 of pseudopregnancy). Embryos were transferred through
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the wall of the fallopian tube as described previously [18], and the
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numbers of offspring were recorded after 19 days.
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Statistics
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Statistical analysis was performed using Prism version 5.0 (GraphPad
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Software, San Diego, CA.). The results are expressed as the mean ±
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standard deviation (SD). The results from each group were compared
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using analysis of variance after arcsine transformation of the percentages;
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P < 0.05 was considered statistically significant.
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RESULTS
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Response to superovulation by IASe in inbred and outbred mice
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strains
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The IASe treatment increased the number of ovulated oocytes with
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higher efficiency than the eCG treatment in all strains of inbred mice
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tested (Table 1); however, the responsiveness to IASe was different among
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the strains (A/J, FVB, CD1 < DBA/2J < C3H/HeJ < BALB/cByJ).
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BALB/cByJ mice ovulated the highest number of IASe-derived oocytes
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(eCG: 29.3±8.9 vs. IASe: 90.3±14.5). A/J mice ovulated the lowest
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number of both eCG- and IASe-derived oocytes, but the IASe treatment
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still produced three times more oocytes than the eCG treatment (eCG:
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7.2±2.7 vs. IASe: 25.5±8.4).
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Fertilization ability of the IASe-derived oocytes in inbred and outbred
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mice strains
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The fertilization rate of IASe-derived oocytes was equal to the
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fertilization rate of eCG-derived oocytes in all strains of mice tested (Table
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2).
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In vitro developmental ability of embryos derived from inbred and
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outbred mice strains ovulated by IASe treatment
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Two-cell embryos derived from the IASe treatment developed
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normally to blastocysts in all strains tested (Table 3). There is no
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significant difference of the developmental rates of the 2-cell embryos to
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four-cell embryos, morulae, or blastocysts between eCG and IASe in all
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groups. More than 90% of 2-cell embryos from C3H/HeJ, DBA/2J, A/J,
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and CD1 developed into blastocysts. Two-cell embryos from FVB/NJ and
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BALB/cByJ had lower developmental rates than those from the other mice
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strains.
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Offspring derived from various inbred mice strains ovulated by IASe
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Two-cell embryos derived from the IASe treatment in all the strains
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developed normally into pups (Table 4). Developmental rates of the 2-cell
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embryos into pups were no significant difference between eCG and IASe.
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The rates varied among the mice strains; CD1 had the highest
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developmental rates and A/J had the lowest developmental rates.
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DISCUSSION
In the present study, we demonstrated IASe-induced superovulation in
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various inbred (A/J, BALB/cByJ, C3HeJ, DBA/2J and FVB/NJ) and
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outbred (CD1) mice strains. The efficacy of IASe on the numbers of
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ovulated oocytes was 1.5 to 3.2 times higher than eCG alone. The
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fertilizing and developmental abilities of the IASe-derived oocytes were
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similar to those of the eCG-derived oocytes. We have shown that
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superovulation using IASe can be used to improve the efficiency of
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producing embryos and pups of genetically engineered mice of various
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inbred and outbred mice strains.
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Reproductive technology is known to be a practical and strategic tool
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to efficiently carry out research using genetically engineered mice [5]. The
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required number of mice can be produced by embryo transfer from fresh
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or cryopreserved embryos derived from IVF; however, if the numbers of
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ovulated oocytes from oocyte donors are low the numbers of embryos or
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animals produced will also be low. Our results suggest that the
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superovulation method using IASe is a very effective way of obtaining
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high numbers of ovulated oocytes to produce embryos and animals in
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mice.
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Since 1956, gonadotropins have been used for superovulation to obtain
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mouse oocytes for experimental purpose [19, 20]. The regimen using eCG
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and hCG (eCG-hCG) has been used widely to induce superovulation in
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mice. Optimization of the eCG-hCG treatment by adjusting age, weight,
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hormone doses, and injection timing was reported to enhance the yield of
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oocytes in various mice strains [6, 21-26]. In 2001, induction of
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superovulation by neutralizing inhibin using IAS was reported in ddY
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mice [27]. Ovulation occurred after a single injection of IAS without hCG
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in ddY mice at the metestrus stage of the estrous cycle. And then, the
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efficacy of IAS confirmed wild-derived strains of mice [28, 29]. In 2015,
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we showed that coadministration of eCG and IAS additively increased the
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number of ovulated oocytes in a female C57BL/6 mouse [11]. Hasegawa
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et al [30] reported the response to superovulation using IAS was enhanced
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by chemically adjusting the metestrus stage using two daily injection of
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progesterone in mature female mice of C57BL/6, BALB/c, ICR, and
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B6D2F1 strains. The adjustment of female status to the metestrus stage
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could help increase the yield of oocytes, but it is not practical because five
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injections (two progesterone injections, two IAS injections, and one hCG
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injection) are required at different times. In this study, we demonstrated
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two injections of IASe and hCG produced sufficient numbers of oocytes in
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various inbred and outbred mice strains at 4 weeks old (Table 1). Higher
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numbers of ovulated oocytes were obtained from all the IASe-treated mice
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strains compared with the numbers reported previously using other
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protocols [5, 6, 30]. It will be interesting to determine whether
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administration of IASe can effectively induce superovulation in other
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strains of mice, other rodents, and mammals.
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strains (Table 1). Previously, Spearow et al reported some major genetic
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differences regulated ovarian responsiveness to gonadotropins and
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gonadotropins-induced ovulation rate among mice strains [31-34]. These
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studies demonstrated C57BL/6 strain was high responder to gonadotropins,
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whereas A/J strain was poor responder to gonadotropins. IASe treatment
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also indicated similar tendency. Folliculogenesis is regulated by the
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function of hypothalamic-pituitary-ovarian axis and some related
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hormones such as kisspeptin, gonadotropin releasing hormone (GnRH),
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FSH, luteinizing hormone (LH), estradiol and progesterone [35]. Further
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experiment should be carried out to elucidate the strain-dependency of the
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responsiveness of gonadotropins or inhibin antiserum.
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The doses of IAS (0.1 mL) and eCG (3.75IU) and the age (4 weeks
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old) of administration were optimized based on our previous study using
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C57BL/6J female mice [11]. Optimal doses of hormones (IAS, eCG or
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hCG) or age may be different among the mouse strains. Therefore,
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optimizing the dose or age will be effective to increase the number of
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ovulated oocytes by IASe method.
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The results of our previous study indicated that IASe-derived oocytes
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had lower fertilization rates than eCG-derived oocytes in C57BL/6 mice
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[11]. We also found that methyl-β-cyclodextrin and reduced glutathione
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improved the fertilization ability of sperm and oocytes in mice [13, 16].
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Methyl-β-cyclodextrin
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cholesterol efflux [36]. Reduced glutathione and cysteine analogs
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enhanced the fertilizing ability of oocytes by breaking disulfide bonds in
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the zona pellucida [16, 37, 38]. In the present study, we found that, after
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induced
sperm
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facilitating
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IVF, the ability of IASe-derived oocytes to be fertilized was similar to that
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of eCG-derived oocytes in all mice strains (Table 2). The reduced
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glutathione treatment may have helped to recover the fertilizing ability of
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IASe-derived oocytes.
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Developmental ability of oocytes obtained by IASe treatment was
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equal to that of eCG treatment in all strains of mice (Table 3 and 4). These
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results suggest that the IASe method is applicable to produce embryos and
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animals of various strains of mice alternative to eCG. Previous studies
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revealed superovulation using the eCG-hCG protocol altered global
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methylation pattern of DNA in oocyte and gene expression at the
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blastocyst stage in mice [39, 40]. Additional experiment should be
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performed to evaluate the quality of IASe-derived oocytes from the point
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of view of epigenetic modifications.
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In the Center for Animal Resources and Development of Kumamoto
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University, we provide a mouse repository service using various mouse
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reproductive techniques [41]. When the superovulation technique using
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IASe was introduced into the mouse repository system, high yields of
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oocytes from oocyte donors were obtained for the purpose of archiving
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cryopreserved embryos and reanimating genetically engineered mice.
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Until now, more than 32,000 strains of genetically engineered mice have
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been archived as livestock, cryopreserved embryos, or cryopreserved
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gametes in mouse repositories [1]. The mouse lines are available via the
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website of the International Mouse Strain Resource, which is an
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international collaboration of mouse repositories that supports researchers
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who use mouse models [42, 43]. The mouse repositories efficiently
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produce, preserve, and supply quality controlled mouse lines to ensure
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reproducible experimental results [44]. The superovulation technique
308
described here will contribute to efficiently archiving cryopreserved
309
embryos of genetically engineered mice using a small number of oocyte
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donors and will produce more live animals for rederivation of the archived
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mouse lines. Improved superovulation techniques will enhance the
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accessibility of genetically engineered mice for the scientific community.
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In summary, we demonstrated IASe-induced superovulation and
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showed that it had higher efficiency of superovulation than eCG-induced
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superovulation in immature female mice of various inbred and outbred
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strains. The quality of oocytes derived from the IASe treatment was
317
similar to that of eCG-derived oocytes. Superovulation using IASe has the
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advantage of maximizing the numbers of ovulated oocyte obtained from
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oocyte donors and minimizing the numbers of oocyte donors required.
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Thus, the efficiency of embryo and animal production is improved and the
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number of animals for oocyte donors is reduced, which meets the 3Rs
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principle (replacement, reduction, and refinement) in animal experiments
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[45]. We strongly believe that the optimized reproductive technology
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using IASe provides good practice for animal experiments using
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genetically engineered mice.
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ACKNOWLEDGEMENTS
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We thank Dr Yoshihisa Hasegawa (Kitasato University, Japan) and Dr
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Gen Watanabe (Tokyo University of Agriculture and Technology, Japan)
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for invaluable advice and discussions concerning IAS. We also thank
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Kiyoko Fukumoto, Tomoko Kondo, Yukie Haruguchi, Yumi Takeshita,
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Yuko Nakamuta, Tomoko Umeno, Shuuji Tsuchiyama, and Ayumi
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Mukunoki for their technical support and helpful discussions.
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Tables
457
TABLE 1 Effect of IASe and eCG treatments on the numbers of
458
ovulated oocytes in inbred and outbred mice strains
459
BALB/cByJ
No. of ovulated oocytes
eCG
20
153
7.7±2.5
IASe
20
497
24.9±8.1*
eCG IASe
C3H/HeJ
eCG IASe
DBA/2J
eCG
FVB/NJ
293
29.3±8.9
10
903
90.3±14.5*
10
286
28.6±8.4
10
520
52.0±18.1*
10
225
22.5±7.8
10
688
68.8±13.6*
eCG
10
168
16.8±3.3
IASe
10
256
25.6±5.9*
eCG
10
202
20.2±8.0
IASe
10
337
33.7±9.9*
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CD1
10
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IASe
Average no. of ovulated oocytes/female
SC
A/J
Superovulation
Total no. of oocyte donors
M AN U
Strains
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456
Average numbers are expressed as the mean ± SD. *P < 0.05 for IASe
461
compared with eCG in each strain.
462 463 464 465 466
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460
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467
TABLE 2 Fertilizing rates of IASe- and eCG-derived oocytes from
468
inbred and outbred mice strains
C3H/HeJ DBA/2J FVB/NJ CD1
472 473 474 475 476
IASe
20
497
eCG
10
293
IASe
10
eCG
136
88.9±15.9
422
84.9±8.5
213
73.4±11.3
903
680
76.4±9.0
10
286
251
87.8±22.6
IASe
10
520
446
85.8±15.5
eCG
10
225
194
86.2±11.0
IASe
10
688
592
86.0±4.5
eCG
10
168
159
94.6±6.7
IASe
10
256
239
93.3±6.6
eCG
10
202
146
72.3±16.0
IASe
10
337
269
79.8±19.6
Fertilization rate is expressed as the mean ± SD.
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153
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470
20
Fertilization rate (%)
SC
BALB/cByJ
eCG
No. of fertilized eggs
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A/J
Superovulation
No. of inseminated oocytes
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Strains
Total no. of oocyte donors
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477
TABLE 3 In vitro developmental ability of embryos derived from
478
IASe treatment in inbred and outbred mice strains
No. of No. of cultured Strains
Superovulation
No. of 4-cell
(%)
2-cell
morulae embryos
DBA/2J
FVB/NJ
100.0±0
IASe
100
97
97.0±2.5
eCG
100
94
94.0±8.7
IASe
100
eCG
482 483 484 485 486
90.3±15.7
95
95.0±0
70
70.0±2.5
90
90.0±5.2
78
78.0±2.9
83
83.0±2.9
68
68.0±6.3
100
100
100.0±0
100
100.0±0
93
93.0±6.3
IASe
100
98
98.0±2.5
97
97.0±5.2
91
91.0±1.4
eCG
100
96
96.0±1.4
92
92.0±4.3
35
35.0±2.5
IASe
100
95
95.0±7.2
86
86.0±6.6
29
29.0±7.5
eCG
100
98
98.0±2.5
95
95.0±0
91
91.0±4.3
eCG
100
100
100.0±0
100
100.0±0
98
98.0±2.5
100
98
98.0±2.5
97
97.0±1.4
94
94.0±1.4
100
99
99.0±1.4
99
99.0±1.4
97
97.0±1.4
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Percentages are expressed as the mean ± SD (n = 3–4).
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481
28
85.0±0
IASe
480
96.8±2.5
85
IASe CD1
30
SC
31
(%)
blastocysts
M AN U
C3H/HeJ
31
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BALB/cByJ
eCG
No. of
(%)
embryos A/J
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479
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487
TABLE 4 In vivo developmental ability of embryos derived from IASe
488
treatment in inbred and outbred mice strains
BALB/cByJ C3H/HeJ
No. of offspring
(%)
eCG
60
10
16.7±7.6
IASe
60
6
10.0±0
eCG
60
16
26.7±7.6
IASe
60
16
26.7±2.9
60
21
35.0±5.0
60
16
26.7±5.8
60
23
38.3±10.4
60
19
31.7±5.8
60
26
43.3±5.8
60
27
45.0±8.7
eCG
60
30
50.0±8.7
IASe
60
31
51.7±2.9
eCG IASe
DBA/2J
eCG IASe
FVB/NJ
eCG
CD1
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Percentages are expressed as the mean ± SD (n = 3).
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490
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IASe
SC
A/J
Superovulation
No. of transferred 2-cell embryos
M AN U
Strains
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489
S0093-691X(16)30119-4
DOI:
10.1016/j.theriogenology.2016.04.076
Reference:
THE 13656
To appear in:
Theriogenology
Received Date: 3 February 2016 Revised Date:
25 April 2016
Accepted Date: 25 April 2016
Please cite this article as: Takeo T, Nakagata N, Immunotherapy using inhibin antiserum enhanced the efficacy of equine chorionic gonadotropin on superovulation in major inbred and outbred mice strains, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.04.076. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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New superovulation technique Inhibin antiserum + Equine chorionic gonadotropin
Inbred mice: A/J, BALB/cByJ, C3HeJ, DBA/2J, FVB/NJ outbred mouse: CD1
♀
No. of ovulated oocytes: 7-29 oocytes
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Conventional superovulation technique Equine chorionic gonadotropin
No. of ovulated oocytes: 24-90 oocytes
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Inhibin antiserum improves the superovulation technique. Coadministration of inhibin antiserum and gonadotropin produces many oocytes. The effect is effective to major inbred and outbred mouse strains. The oocytes normally fertilize and develop to offspring.
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Immunotherapy using inhibin antiserum enhanced the efficacy of
2
equine chorionic gonadotropin on superovulation in major inbred and
3
outbred mice strains
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1
4
Running title: Improvement of superovulation by inhibin antiserum
6
Summary sentence: Coadministration of inhibin antiserum and equine
7
chorionic gonadotropin improves superovulation technique and is very
8
effective in obtaining high number of ovulated oocytes in major inbred
9
and outbred mice strains.
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Key words: Superovulation, inhibin antiserum, equine chorionic
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gonadotropin, oocytes, mice, embryo production, mouse strain, mouse
12
repository
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Toru Takeo and Naomi Nakagata
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Division of Reproductive Engineering, Center for Animal Resources and
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Development (CARD), Kumamoto University, Chuo-ku, Kumamoto,
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Japan
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Correspondence:
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Naomi Nakagata, E-mail: [email protected], Telephone:
22
+81-96-373-6570, Fax: +81-373-6566
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ABSTRACT
25
Improvement of the superovulation technique will help to enhance the
26
efficiency of embryo and animal production. Blocking inhibin using
27
inhibin antiserum (IAS) is known to promote follicular development by
28
increasing the level of follicular stimulating hormone. Previously, we
29
reported that coadministration of IAS and equine chorionic gonadotropin
30
(eCG) produced more than 100 oocytes from a single female C57BL/6
31
mouse at 4 weeks old. The oocytes derived from the IAS + eCG (IASe)
32
treatment were able to fertilize and develop normally into offspring. In this
33
study, we examined the effect of IASe treatment on the numbers of
34
ovulated oocytes in major inbred (A/J, BALB/cByJ, C3HeJ, DBA/2J, and
35
FVB/NJ) and outbred (CD1) mice strains at 4 weeks old. We confirmed
36
the fertilization and developmental ability of the IASe-derived oocytes.
37
IASe treatment ovulated 1.5 to 3.2 times higher numbers of oocytes than
38
eCG treatment alone. The fertilization rate of IASe-derived oocytes was
39
similar to that of eCG-derived oocytes. In vitro and in vivo developmental
40
rates of the embryos derived from IASe was similar to the rates of
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embryos derived from eCG. We have shown that superovulation by IASe
42
is very effective in obtaining high numbers of ovulated oocytes from small
43
numbers of oocyte donor in a number of mice strains. The superovulation
44
technique will contribute to the archiving of cryopreserved embryos of
45
genetically engineered mice using small numbers of donors and has the
46
potential to produce more live animals for rederivation of the archived
47
mouse lines in mouse repositories.
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INTRODUCTION
Mouse repositories that support the production, preservation, and
51
distribution of genetically engineered mice have been established to
52
improve the accessibility of these mice to the scientific community [1].
53
The archived lines of genetically engineered mice are published by the
54
International Mouse Strain Resource (IMSR; http://www.findmice.org/).
55
Researchers can order and obtain mouse lines via the IMSR.
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Reproductive technology is a powerful tool to efficiently manage
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genetically engineered mice in mouse repositories [2]. In vitro fertilization
58
(IVF) using sperm and oocytes harvested from genetically engineered
59
mice can readily produce a huge number of embryos. The embryos can be
60
transferred to recipients to obtain the numbers of offspring required for a
4
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planned animal experiment or they can be preserved in liquid nitrogen for
62
later use. The application of reproductive techniques for animal production
63
allows the precise management of size and schedule of experiments using
64
genetically engineered mice.
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The number of ovulated oocytes is a bottleneck that impacts the
66
numbers of embryos and animals that can be produced by reproductive
67
technology. It is important, therefore, to increase the number of ovulated
68
oocytes using superovulation techniques, which artificially promote
69
folliculogenesis
70
Superovulation has been used successfully to obtain oocytes for the
71
production of genetically engineered mice [3]. Traditionally, a regimen of
72
equine chorionic gonadotropin (eCG) and human chorionic gonadotropin
73
(hCG) has been used to induce superovulation in mice [4]; however, the
74
response to this technique is known to vary among inbred and hybrid
75
mouse strains [5, 6].
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induce
ovulation,
by
hormone
treatment.
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SC
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Administration of inhibin antiserum (IAS) has been shown to induce
77
superovulation in several species [7]. IAS neutralized inhibin, which is a
78
non-steroidal hormone that acts on pituitary cells to suppress the
79
production of follicle-stimulating hormone (FSH) [8]. The blocking of
80
inhibin by IAS promoted follicular development by facilitating the
5
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production of FSH [9, 10]. Recently, we developed a novel superovulation
82
technique by the coadministration of IAS and eCG [11], which yielded
83
more than 100 oocytes from a single female C57BL/6 mouse at 4 weeks
84
old. The ovulated oocytes were fertilized in vitro and live pups developed
85
after IVF and embryo transfer [11]. However, the efficacy of the IAS +
86
eCG (IASe) treatment on female mice of major inbred and outbred strains
87
has not been examined so far.
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In this study, we investigated the effect of IASe treatment on the
89
number of ovulated oocytes in major inbred and outbred mouse strains of
90
A/J, BALB/c, C3H, CD1, DBA, and FVB mice. The ovulated oocytes
91
were used for IVF, and the embryos were cultured and transferred to
92
confirm their fertilization and developmental ability.
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MATERIALS AND METHODS
95
Animals
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Inbred mouse strains of A/J (SLC, Shizuoka, Japan), BALB/cByJ
97
(CLEA Japan, Tokyo, Japan), C3HeJ (CLEA Japan), DBA/2J (CLEA
98
Japan), and FVB/NJ (CLEA Japan) and an outbred mouse strain of CD1
99
(Charles River Japan, Yokohama, Japan) were purchased and used as
100
donors of sperm (from male mice at 12–15 weeks old) and oocytes (from
6
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female mice at 4 weeks old). ICR mice (CLEA Japan) at 8–16 weeks old
102
were used as recipients of 2-cell embryos. All the mice were housed in a
103
specific-pathogen-free room under a 12-h dark-light cycle (light from
104
07:00 to 19:00) at a 22 ± 1°C with ad libitum food and water. The Animal
105
Care and Use Committee of the Kumamoto University School of Medicine
106
approved the protocols for the experiments using animals. The mice were
107
properly handled and euthanized by the person who was admitted and
108
directed by the Animal Care and Use Committee of Kumamoto University.
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109 110
Medium
IAS was prepared as described previously and preserved at -20oC
112
before use [9, 10]. Sperm were preincubated in a modified Krebs–Ringer
113
bicarbonate solution (TYH) containing 1.0 mg/mL polyvinyl alcohol
114
(Sigma) and 0.75 mM methyl-β-cyclodextrin (Sigma) as sperm
115
preincubation medium [12, 13]. Calcium-enhanced human tubal fluid
116
(mHTF) containing 0.25 mM reduced glutathione (Sigma) was used as
117
fertilization medium [14-16]. Potassium simplex optimized medium
118
(KSOM) was used to handle and culture the 2-cell embryos to the
119
blastocyst stage [17]. All media were prepared and stored at 4oC no longer
120
than 3 months before use. Sperm preincubation medium, fertilization
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medium and embryo culture medium were preincubated in CO2 incubator
122
(5% CO2 and 95% air) at 37oC for 30 min before use.
124
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Superovulation
Superovulation was performed as described in our previous study [11].
126
Briefly, immature female mice at 4 weeks old were administered 7.5 IU
127
eCG (ASKA Pharmaceutical Co. Ltd, Japan) or IASe (mixed solution of
128
0.1 mL IAS and 3.75 IU eCG) intraperitoneally. Forty-eight hours after the
129
injection of eCG or IASe, the mice were administered 7.5 IU hCG (ASKA
130
Pharmaceutical Co. Ltd, Japan) intraperitoneally. At 17 h after hCG
131
administration, the mice were euthanized by cervical dislocation and the
132
oviducts were collected quickly and transferred to a fertilization dish
133
covered with paraffin oil. Under microscopic observation, cumulus–oocyte
134
complexes were collected from the oviducts by dissecting needle and
135
transferred to a 200-µL drop of fertilization medium. The numbers of
136
ovulated oocytes and their ability to be fertilized were examined in each
137
group.
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138 139 140
In vitro fertilization Male mice were euthanized by cervical dislocation and the cauda
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epididymides were collected and transferred to a dish of sperm
142
preincubation medium covered with paraffin oil. Aliquots of sperm were
143
collected from the cauda epididymides using a dissecting needle and
144
transferred to a 100-µL drop of sperm preincubation medium. Sperm were
145
preincubated for 60 min to induce capacitation and then added to the
146
fertilization drop containing the cumulus–oocyte complexes and cultured
147
with oocytes at 37°C in an atmosphere containing 5% CO2 for 3 h. The
148
concentration of motile sperm was measured by computer-assisted sperm
149
analysis (IVOS, Hamilton Thorn Inc.) before insemination and then
150
adjusted to 400–800 motile sperm/µL in fertilization medium. At 3 h after
151
insemination, oocytes were washed in three drops of mHTF (80 µL) and
152
the number of ovulated oocytes was examined. At 24 h after insemination,
153
fertilization rates were calculated as the total number of 2-cell embryos
154
divided by the total number of inseminated oocytes multiplied by 100.
156 157
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Embryo culture and transfer To evaluate the development of the 2-cell embryos produced by IVF,
158
we performed embryo culture and transfer. After IVF, the 2-cell embryos
159
were divided into two groups; one that was cultured to the blastocyst stage
160
in a 100-µL drop of KSOM and another that was transferred into the
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oviducts of ICR females (10 embryos/oviduct) on the day a vaginal plug
162
was found (day 1 of pseudopregnancy). Embryos were transferred through
163
the wall of the fallopian tube as described previously [18], and the
164
numbers of offspring were recorded after 19 days.
165
Statistics
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166
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Statistical analysis was performed using Prism version 5.0 (GraphPad
168
Software, San Diego, CA.). The results are expressed as the mean ±
169
standard deviation (SD). The results from each group were compared
170
using analysis of variance after arcsine transformation of the percentages;
171
P < 0.05 was considered statistically significant.
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RESULTS
174
Response to superovulation by IASe in inbred and outbred mice
175
strains
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The IASe treatment increased the number of ovulated oocytes with
177
higher efficiency than the eCG treatment in all strains of inbred mice
178
tested (Table 1); however, the responsiveness to IASe was different among
179
the strains (A/J, FVB, CD1 < DBA/2J < C3H/HeJ < BALB/cByJ).
180
BALB/cByJ mice ovulated the highest number of IASe-derived oocytes
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(eCG: 29.3±8.9 vs. IASe: 90.3±14.5). A/J mice ovulated the lowest
182
number of both eCG- and IASe-derived oocytes, but the IASe treatment
183
still produced three times more oocytes than the eCG treatment (eCG:
184
7.2±2.7 vs. IASe: 25.5±8.4).
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185
Fertilization ability of the IASe-derived oocytes in inbred and outbred
187
mice strains
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The fertilization rate of IASe-derived oocytes was equal to the
189
fertilization rate of eCG-derived oocytes in all strains of mice tested (Table
190
2).
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In vitro developmental ability of embryos derived from inbred and
193
outbred mice strains ovulated by IASe treatment
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Two-cell embryos derived from the IASe treatment developed
195
normally to blastocysts in all strains tested (Table 3). There is no
196
significant difference of the developmental rates of the 2-cell embryos to
197
four-cell embryos, morulae, or blastocysts between eCG and IASe in all
198
groups. More than 90% of 2-cell embryos from C3H/HeJ, DBA/2J, A/J,
199
and CD1 developed into blastocysts. Two-cell embryos from FVB/NJ and
200
BALB/cByJ had lower developmental rates than those from the other mice
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201
strains.
202
Offspring derived from various inbred mice strains ovulated by IASe
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Two-cell embryos derived from the IASe treatment in all the strains
205
developed normally into pups (Table 4). Developmental rates of the 2-cell
206
embryos into pups were no significant difference between eCG and IASe.
207
The rates varied among the mice strains; CD1 had the highest
208
developmental rates and A/J had the lowest developmental rates.
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209 210
DISCUSSION
In the present study, we demonstrated IASe-induced superovulation in
212
various inbred (A/J, BALB/cByJ, C3HeJ, DBA/2J and FVB/NJ) and
213
outbred (CD1) mice strains. The efficacy of IASe on the numbers of
214
ovulated oocytes was 1.5 to 3.2 times higher than eCG alone. The
215
fertilizing and developmental abilities of the IASe-derived oocytes were
216
similar to those of the eCG-derived oocytes. We have shown that
217
superovulation using IASe can be used to improve the efficiency of
218
producing embryos and pups of genetically engineered mice of various
219
inbred and outbred mice strains.
220
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Reproductive technology is known to be a practical and strategic tool
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to efficiently carry out research using genetically engineered mice [5]. The
222
required number of mice can be produced by embryo transfer from fresh
223
or cryopreserved embryos derived from IVF; however, if the numbers of
224
ovulated oocytes from oocyte donors are low the numbers of embryos or
225
animals produced will also be low. Our results suggest that the
226
superovulation method using IASe is a very effective way of obtaining
227
high numbers of ovulated oocytes to produce embryos and animals in
228
mice.
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Since 1956, gonadotropins have been used for superovulation to obtain
230
mouse oocytes for experimental purpose [19, 20]. The regimen using eCG
231
and hCG (eCG-hCG) has been used widely to induce superovulation in
232
mice. Optimization of the eCG-hCG treatment by adjusting age, weight,
233
hormone doses, and injection timing was reported to enhance the yield of
234
oocytes in various mice strains [6, 21-26]. In 2001, induction of
235
superovulation by neutralizing inhibin using IAS was reported in ddY
236
mice [27]. Ovulation occurred after a single injection of IAS without hCG
237
in ddY mice at the metestrus stage of the estrous cycle. And then, the
238
efficacy of IAS confirmed wild-derived strains of mice [28, 29]. In 2015,
239
we showed that coadministration of eCG and IAS additively increased the
240
number of ovulated oocytes in a female C57BL/6 mouse [11]. Hasegawa
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et al [30] reported the response to superovulation using IAS was enhanced
242
by chemically adjusting the metestrus stage using two daily injection of
243
progesterone in mature female mice of C57BL/6, BALB/c, ICR, and
244
B6D2F1 strains. The adjustment of female status to the metestrus stage
245
could help increase the yield of oocytes, but it is not practical because five
246
injections (two progesterone injections, two IAS injections, and one hCG
247
injection) are required at different times. In this study, we demonstrated
248
two injections of IASe and hCG produced sufficient numbers of oocytes in
249
various inbred and outbred mice strains at 4 weeks old (Table 1). Higher
250
numbers of ovulated oocytes were obtained from all the IASe-treated mice
251
strains compared with the numbers reported previously using other
252
protocols [5, 6, 30]. It will be interesting to determine whether
253
administration of IASe can effectively induce superovulation in other
254
strains of mice, other rodents, and mammals.
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There was the difference of responsiveness to IASe among mice
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256
strains (Table 1). Previously, Spearow et al reported some major genetic
257
differences regulated ovarian responsiveness to gonadotropins and
258
gonadotropins-induced ovulation rate among mice strains [31-34]. These
259
studies demonstrated C57BL/6 strain was high responder to gonadotropins,
260
whereas A/J strain was poor responder to gonadotropins. IASe treatment
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also indicated similar tendency. Folliculogenesis is regulated by the
262
function of hypothalamic-pituitary-ovarian axis and some related
263
hormones such as kisspeptin, gonadotropin releasing hormone (GnRH),
264
FSH, luteinizing hormone (LH), estradiol and progesterone [35]. Further
265
experiment should be carried out to elucidate the strain-dependency of the
266
responsiveness of gonadotropins or inhibin antiserum.
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The doses of IAS (0.1 mL) and eCG (3.75IU) and the age (4 weeks
268
old) of administration were optimized based on our previous study using
269
C57BL/6J female mice [11]. Optimal doses of hormones (IAS, eCG or
270
hCG) or age may be different among the mouse strains. Therefore,
271
optimizing the dose or age will be effective to increase the number of
272
ovulated oocytes by IASe method.
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The results of our previous study indicated that IASe-derived oocytes
274
had lower fertilization rates than eCG-derived oocytes in C57BL/6 mice
275
[11]. We also found that methyl-β-cyclodextrin and reduced glutathione
276
improved the fertilization ability of sperm and oocytes in mice [13, 16].
277
Methyl-β-cyclodextrin
278
cholesterol efflux [36]. Reduced glutathione and cysteine analogs
279
enhanced the fertilizing ability of oocytes by breaking disulfide bonds in
280
the zona pellucida [16, 37, 38]. In the present study, we found that, after
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induced
sperm
capacitation
by
facilitating
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IVF, the ability of IASe-derived oocytes to be fertilized was similar to that
282
of eCG-derived oocytes in all mice strains (Table 2). The reduced
283
glutathione treatment may have helped to recover the fertilizing ability of
284
IASe-derived oocytes.
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Developmental ability of oocytes obtained by IASe treatment was
286
equal to that of eCG treatment in all strains of mice (Table 3 and 4). These
287
results suggest that the IASe method is applicable to produce embryos and
288
animals of various strains of mice alternative to eCG. Previous studies
289
revealed superovulation using the eCG-hCG protocol altered global
290
methylation pattern of DNA in oocyte and gene expression at the
291
blastocyst stage in mice [39, 40]. Additional experiment should be
292
performed to evaluate the quality of IASe-derived oocytes from the point
293
of view of epigenetic modifications.
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In the Center for Animal Resources and Development of Kumamoto
295
University, we provide a mouse repository service using various mouse
296
reproductive techniques [41]. When the superovulation technique using
297
IASe was introduced into the mouse repository system, high yields of
298
oocytes from oocyte donors were obtained for the purpose of archiving
299
cryopreserved embryos and reanimating genetically engineered mice.
300
Until now, more than 32,000 strains of genetically engineered mice have
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been archived as livestock, cryopreserved embryos, or cryopreserved
302
gametes in mouse repositories [1]. The mouse lines are available via the
303
website of the International Mouse Strain Resource, which is an
304
international collaboration of mouse repositories that supports researchers
305
who use mouse models [42, 43]. The mouse repositories efficiently
306
produce, preserve, and supply quality controlled mouse lines to ensure
307
reproducible experimental results [44]. The superovulation technique
308
described here will contribute to efficiently archiving cryopreserved
309
embryos of genetically engineered mice using a small number of oocyte
310
donors and will produce more live animals for rederivation of the archived
311
mouse lines. Improved superovulation techniques will enhance the
312
accessibility of genetically engineered mice for the scientific community.
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In summary, we demonstrated IASe-induced superovulation and
314
showed that it had higher efficiency of superovulation than eCG-induced
315
superovulation in immature female mice of various inbred and outbred
316
strains. The quality of oocytes derived from the IASe treatment was
317
similar to that of eCG-derived oocytes. Superovulation using IASe has the
318
advantage of maximizing the numbers of ovulated oocyte obtained from
319
oocyte donors and minimizing the numbers of oocyte donors required.
320
Thus, the efficiency of embryo and animal production is improved and the
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number of animals for oocyte donors is reduced, which meets the 3Rs
322
principle (replacement, reduction, and refinement) in animal experiments
323
[45]. We strongly believe that the optimized reproductive technology
324
using IASe provides good practice for animal experiments using
325
genetically engineered mice.
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ACKNOWLEDGEMENTS
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We thank Dr Yoshihisa Hasegawa (Kitasato University, Japan) and Dr
329
Gen Watanabe (Tokyo University of Agriculture and Technology, Japan)
330
for invaluable advice and discussions concerning IAS. We also thank
331
Kiyoko Fukumoto, Tomoko Kondo, Yukie Haruguchi, Yumi Takeshita,
332
Yuko Nakamuta, Tomoko Umeno, Shuuji Tsuchiyama, and Ayumi
333
Mukunoki for their technical support and helpful discussions.
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Tables
457
TABLE 1 Effect of IASe and eCG treatments on the numbers of
458
ovulated oocytes in inbred and outbred mice strains
459
BALB/cByJ
No. of ovulated oocytes
eCG
20
153
7.7±2.5
IASe
20
497
24.9±8.1*
eCG IASe
C3H/HeJ
eCG IASe
DBA/2J
eCG
FVB/NJ
293
29.3±8.9
10
903
90.3±14.5*
10
286
28.6±8.4
10
520
52.0±18.1*
10
225
22.5±7.8
10
688
68.8±13.6*
eCG
10
168
16.8±3.3
IASe
10
256
25.6±5.9*
eCG
10
202
20.2±8.0
IASe
10
337
33.7±9.9*
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CD1
10
TE D
IASe
Average no. of ovulated oocytes/female
SC
A/J
Superovulation
Total no. of oocyte donors
M AN U
Strains
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456
Average numbers are expressed as the mean ± SD. *P < 0.05 for IASe
461
compared with eCG in each strain.
462 463 464 465 466
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467
TABLE 2 Fertilizing rates of IASe- and eCG-derived oocytes from
468
inbred and outbred mice strains
C3H/HeJ DBA/2J FVB/NJ CD1
472 473 474 475 476
IASe
20
497
eCG
10
293
IASe
10
eCG
136
88.9±15.9
422
84.9±8.5
213
73.4±11.3
903
680
76.4±9.0
10
286
251
87.8±22.6
IASe
10
520
446
85.8±15.5
eCG
10
225
194
86.2±11.0
IASe
10
688
592
86.0±4.5
eCG
10
168
159
94.6±6.7
IASe
10
256
239
93.3±6.6
eCG
10
202
146
72.3±16.0
IASe
10
337
269
79.8±19.6
Fertilization rate is expressed as the mean ± SD.
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20
Fertilization rate (%)
SC
BALB/cByJ
eCG
No. of fertilized eggs
M AN U
A/J
Superovulation
No. of inseminated oocytes
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Strains
Total no. of oocyte donors
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469
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477
TABLE 3 In vitro developmental ability of embryos derived from
478
IASe treatment in inbred and outbred mice strains
No. of No. of cultured Strains
Superovulation
No. of 4-cell
(%)
2-cell
morulae embryos
DBA/2J
FVB/NJ
100.0±0
IASe
100
97
97.0±2.5
eCG
100
94
94.0±8.7
IASe
100
eCG
482 483 484 485 486
90.3±15.7
95
95.0±0
70
70.0±2.5
90
90.0±5.2
78
78.0±2.9
83
83.0±2.9
68
68.0±6.3
100
100
100.0±0
100
100.0±0
93
93.0±6.3
IASe
100
98
98.0±2.5
97
97.0±5.2
91
91.0±1.4
eCG
100
96
96.0±1.4
92
92.0±4.3
35
35.0±2.5
IASe
100
95
95.0±7.2
86
86.0±6.6
29
29.0±7.5
eCG
100
98
98.0±2.5
95
95.0±0
91
91.0±4.3
eCG
100
100
100.0±0
100
100.0±0
98
98.0±2.5
100
98
98.0±2.5
97
97.0±1.4
94
94.0±1.4
100
99
99.0±1.4
99
99.0±1.4
97
97.0±1.4
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Percentages are expressed as the mean ± SD (n = 3–4).
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481
28
85.0±0
IASe
480
96.8±2.5
85
IASe CD1
30
SC
31
(%)
blastocysts
M AN U
C3H/HeJ
31
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BALB/cByJ
eCG
No. of
(%)
embryos A/J
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479
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487
TABLE 4 In vivo developmental ability of embryos derived from IASe
488
treatment in inbred and outbred mice strains
BALB/cByJ C3H/HeJ
No. of offspring
(%)
eCG
60
10
16.7±7.6
IASe
60
6
10.0±0
eCG
60
16
26.7±7.6
IASe
60
16
26.7±2.9
60
21
35.0±5.0
60
16
26.7±5.8
60
23
38.3±10.4
60
19
31.7±5.8
60
26
43.3±5.8
60
27
45.0±8.7
eCG
60
30
50.0±8.7
IASe
60
31
51.7±2.9
eCG IASe
DBA/2J
eCG IASe
FVB/NJ
eCG
CD1
EP
Percentages are expressed as the mean ± SD (n = 3).
AC C
490
TE D
IASe
SC
A/J
Superovulation
No. of transferred 2-cell embryos
M AN U
Strains
RI PT
489