grafting. However, it must be noted that establishment of proper
hypothalamic/pituitary/gonadal/adrenal axis signaling network
may take several months depending on the OT donor species
[22]. For laboratory rodents, restoration of the reproductive cycle
can be monitored 3–4 weeks post-grafting.
3.9 Vaginal Smears
1. Transfer 10 mL warm physiologic saline solution in 15 mL test
tube, and prepare fire-polished pasture pipettes for each animal.
2. Draw approximately 30–50 μL saline solution in the tip of the
pasture pipette using a rubber bulb, and restrain the animal by
holding from the base of the tail.
3. Gently insert the tip of the pasture pipette in the vagina, and
wash few times while still inside, and finally suck the content
and expel onto a microscope slide, and cover with a cover slip
for immediate evaluation under 10Â objective (see Notes 9 and
10).
4. Alternatively, air dry the slides completely, plunge into a jar
containing methanol for 5 min to fix before staining with
methylene blue for 2 min, and wash the slide twice in saline
solution. After completely drying, examine the slides immediately or later.
3.10 Vaginal
Cytology
and Corresponding
Estrus Cycle Stages
The reestablishment of the reproductive cycle of the recipients can
be determined by monitoring vaginal cytology as early as 4 weeks
after transplantation surgery. To produce live pups after orthotopic
OT grafting, the OT recipients displaying regular reproductive
cycle (4–5 days) can be mated with appropriate proven breeder
male. It is expected that the recipients become pregnant within
3 months after transplantation and produce live pups. Depending
on the strain, the OT recipients are able to produce 3–6 live pups
per litter (see Fig. 3f).
The complete estrous cycles in laboratory mice and rats occur
over 4 or 5 days and can be divided into four stages: proestrus,
estrus, metestrus, and diestrus. The duration of proestrus phase is
about 0.5 days and corresponds to the preovulatory stage with
increase in serum estradiol level before the preovulatory LH surge
and subsequent ovulation (see Fig. 4a). The microscope field is
predominantly covered with nucleated epithelial cells individually
or in clusters. Occasionally, a few cornified cells may be observed.
The duration of estrus phase is also about 0.5 days and corresponds
with the presence of cornified squamous epithelial cells mostly in
clusters. These cells have irregular edges and have no visible nucleus
(see Fig. 4b). Serum estradiol remains high throughout the morning and declines back to basal levels in the afternoon. The duration
of metestrus phase is about 1 day and marked with the presence of
mixture of cell types including mostly leucocytes, a few nucleated
Rodent Ovarian Tissue Cryopreservation
479
hypothalamic/pituitary/gonadal/adrenal axis signaling network
may take several months depending on the OT donor species
[22]. For laboratory rodents, restoration of the reproductive cycle
can be monitored 3–4 weeks post-grafting.
3.9 Vaginal Smears
1. Transfer 10 mL warm physiologic saline solution in 15 mL test
tube, and prepare fire-polished pasture pipettes for each animal.
2. Draw approximately 30–50 μL saline solution in the tip of the
pasture pipette using a rubber bulb, and restrain the animal by
holding from the base of the tail.
3. Gently insert the tip of the pasture pipette in the vagina, and
wash few times while still inside, and finally suck the content
and expel onto a microscope slide, and cover with a cover slip
for immediate evaluation under 10Â objective (see Notes 9 and
10).
4. Alternatively, air dry the slides completely, plunge into a jar
containing methanol for 5 min to fix before staining with
methylene blue for 2 min, and wash the slide twice in saline
solution. After completely drying, examine the slides immediately or later.
3.10 Vaginal
Cytology
and Corresponding
Estrus Cycle Stages
The reestablishment of the reproductive cycle of the recipients can
be determined by monitoring vaginal cytology as early as 4 weeks
after transplantation surgery. To produce live pups after orthotopic
OT grafting, the OT recipients displaying regular reproductive
cycle (4–5 days) can be mated with appropriate proven breeder
male. It is expected that the recipients become pregnant within
3 months after transplantation and produce live pups. Depending
on the strain, the OT recipients are able to produce 3–6 live pups
per litter (see Fig. 3f).
The complete estrous cycles in laboratory mice and rats occur
over 4 or 5 days and can be divided into four stages: proestrus,
estrus, metestrus, and diestrus. The duration of proestrus phase is
about 0.5 days and corresponds to the preovulatory stage with
increase in serum estradiol level before the preovulatory LH surge
and subsequent ovulation (see Fig. 4a). The microscope field is
predominantly covered with nucleated epithelial cells individually
or in clusters. Occasionally, a few cornified cells may be observed.
The duration of estrus phase is also about 0.5 days and corresponds
with the presence of cornified squamous epithelial cells mostly in
clusters. These cells have irregular edges and have no visible nucleus
(see Fig. 4b). Serum estradiol remains high throughout the morning and declines back to basal levels in the afternoon. The duration
of metestrus phase is about 1 day and marked with the presence of
mixture of cell types including mostly leucocytes, a few nucleated
Rodent Ovarian Tissue Cryopreservation
479