Proestrus

All experimental protocols for the procedures with rats were pre-approved by the Washington State University Animal Care and Use Committee (IACUC approval # 02568-026). The University Department of Environmental Health and Safety approved all the protocols for the use of hazardous chemicals in this experiment. Sprague Dawley SD female and male rats of an outbred strain (Harlan) at about 70 and 100 days of age were maintained in ventilated (up to 50 air exchanges/hour) isolator cages (cages with dimensions of 10 ¾″ W×19 ¼″ D×10 ¾″ H, 143 square inch floor space, fitted in Micro-vent 36-cage rat racks; Allentown Inc., Allentown, NJ) containing Aspen Sani chips (pinewood shavings from Harlan) as bedding, and a 14 h light: 10 h dark regimen, at a temperature of 70 F and humidity of 25% to 35%. The mean light intensity in the animal rooms ranged from 22 to 26 ft-candles. Rats were fed ad lib with standard rat diet (8640 Teklad 22/5 Rodent Diet; Harlan) and ad lib tap water for drinking. During the procedures, rats were held in an animal transfer station (AniGard 6VF, The Baker Company, Sanford, ME) that provided an air velocity of about 0.5 inch.
At proestrus as determined by daily vaginal smears, the female rats, (90 days) were pair-mated with male rats (120 days). On the next day, the females were separated and their vaginal smears were examined microscopically and if they were sperm-positive (day 0) the rats were tentatively considered pregnant and then weighed with a digital animal weighing balance to monitor increases in body weight. Vaginal smears were continued for monitoring diestrus status in these rats until day 7. On embryonic day 7 (E-7) these females were weighed to determine if there was a significant increase in (greater than about 10 g) body weight, to confirm pregnancy in sperm-positive females. These pregnant rats were then given daily intraperitoneal injections of any one of the following single chemicals or mixtures with an equal volume of sesame oil (Sigma) on days E-8 through E-14 of gestation [43] (link). Treatment groups were Control, Pesticide (Permethrin+DEET), Plastics (Bisphenol-A, DBP and DEHP), Dioxin (TCDD), and Jet Fuel (JP8 hydrocarbon). The pregnant female rats treated with various mixtures were designated as the F0 generation. When there was a drop in the litter size and the sex ratio of pups in F1 generation of Plastics group, another treatment group was included with only half the dose of Bisphenol-A, DBP and DEHP and this group was designated ‘Low Dose Plastics’ group. Doses, percent of oral LD50, and sources of chemicals for the compounds are given in Table S1A.

The estrous cycle identification tool was developed using qualitative data from the literature [5] , [9] (link), [10] for the proportion of each cell type in a smear. A graphical representation of the existing data was created to represent the typical changes in cell types that occur during the entire estrous cycle. The continuous changes in cell types (leukocytes, nucleated epithelial, and cornified epithelial) occurring during the estrous cycle result in the lack of clear demarcations between stages and can make it difficult to determine the stage of the estrous cycle. For example, the vaginal cytology of a mouse in estrus is characterized by many cornified epithelia cells. However, if the mouse is in early estrus, nucleated epithelial cells may also be present. Presented here (Figure 1) is an estrous cycle identification tool that shows the changes in cell populations during the entire cycle. The estrous cycle identification tool makes it clear what cells types are present at each point of the cycle, including the transitional phases between each stage.
The estrous cycle identification tool is a visual aid that shows the 4 estrous stages and the relative proportion of cells present in each stage. Each cell type is shown in a different color. The name of each stage of the estrous cycle is shown on the outside of the circle progressing clockwise from one stage to the next. The 4 quadrants are different sizes to represent a rough estimate of how much time is spent in each stage of the estrous cycle.
To use the estrous cycle identification tool, collect cells using the vaginal cytology method described and view them using a compound microscope. Identify the cell types present on the slide and note the relative proportion of each cell type. For example, there may be all leukocytes on the slide or there may be about half cornified epithelial and about half nucleated epithelial cells. Next, look at the estrous cycle identification tool (Figure 1) and place an imaginary arrow on the chart with the end on the center of the chart like a hand on a clock. The arrow is moved clockwise until the cell types and proportion appear under the arrow. Once the arrow is placed, it points to the corresponding stage of estrous.
This tool makes it easy to determine the stage of the cycle when vaginal cytology is used. The relative amount and type of cells present during early proestrus and late metestrus are similar. The nucleated epithelial cells in proestrus are often well-formed, but are often irregularly shaped and vacuolated in metestrus [3] (link). Alternatively, early proestrus and late metestrus can be distinguished using the visual method.

Control (VEH) and prenatally-androgenized (PNA) male and female mice were generated using the well-characterized prenatally androgenized (PNA) mouse model protocol (29 (link)–33 (link), 57 (link)). Adult male and female C57BL/6J mice were paired overnight on the day of proestrus. Gestational day 1 was recorded as the following day after overnight mating and the male was removed from the cage. Females were then monitored for signs of pregnancy such as increased body weight and increased belly circumference. From gestational day 16-19, pregnant dams received a daily subcutaneous (s.c.) injection in the nape of the neck of either 100 µL dihydrotestosterone (DHT, 250 µg/100µL) in sesame oil as the PNA treatment or 100 µL of sesame oil only as the vehicle control. This window of prenatal androgen exposure has been shown to lead to PCOS-like features in mice and largely avoid the critical period for the differentiation of external genitalia (29 (link)–33 (link), 57 (link)).The male (M) and female (F) offspring of dams injected with DHT (PNAM or PNAF) and vehicle control (VEH) mice were studied from adulthood (postnatal day (PND) 60 onward) in the following experimental protocols. Oestrous cyclicity of VEH and PNA female mice was assessed to establish the expected loss of oestrous cyclicity in PNA mice by collecting daily vaginal smears over a 20-day period (PND 60–80) (Figure S1) as previously described (29 (link)–31 (link), 33 (link), 58 (link)).

The phase of the estrous cycle in females was determined in wet smears immediately after collection (direct cytology, unstained slides) with a Nikon microscope [20 (link),43 (link),44 (link)]. Vaginal swabs were obtained using sterile saline and examined on a slide in a drop of saline. Microscopic examination was performed with a 10× objective to determine the relationship between cell types and a 40× objective to recognize cell types.
The swabs contained four types of cells:
1. Leukocytes (neutrophils or polymorphonuclear cells), which are very small round cells.
2. Small nucleated epithelial cells, which are small non-keratinizing cells of a round or oval shape.
3. Large nucleated epithelial cells of a round or polygonal shape with serrated or angular edges.
4. Non-nuclear keratinized epithelial cells or needle-like cells.
Diestrus (the longest phase) lasted on average 48–72 h and was characterized by the predominance of leukocytes. Leukocytes were absent in the stage of proestrus and estrus. Some leukocytes and epithelial cells were detected during metestrus (6–8 h).