Animal care

All experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and all experiments approved by the Monash University/Monash Medical Centre Animal Ethics Committee. These animals were sourced from our own research colony, where temperature is maintained at 25–27°C, humidity 30–40%, with a 12-hour light-dark cycle (lights on 7:00 am ). Virgin female spiny mice (n = 18) aged 12–16 weeks, were housed in groups of 5–6 per cage. Cages were lined in wooden shavings and enrichment provided in forms of cardboard tunnels and climbing apparatuses. Food (rat and mouse cubes, Specialty Feeds, Glen Forrest, WA) and water were provided ad libitum and supplemental fresh vegetables (carrots and celery, up to 50 g per cage) were provided weekly. Animals were checked at least twice daily and cages were cleaned weekly. Female weights were monitored daily throughout the experiment.

Vaginal lavage

Vaginal lavages were performed daily (n = 18 females) between 12:30 pm and 2:30 pm using sterile saline solution (0.9% wt/vol), and the time of sampling recorded each day. Female spiny mice were restrained in the supine position with a small hand towel by gently scruffing behind the neck and shoulders (a hand towel is necessary due to the fragility of spiny mouse connective tissues and the propensity for skin to easily tear). The vulva was then lubricated with nonscented, water-based lubricant (Lifestyles, Ansell, Melbourne, Australia). A 1-mL plastic transfer pipette was used to draw up ∼50 μL of saline, before gentle insertion into the vaginal canal. The length of the spiny mouse vaginal canal is 16.3 ± 1.1 mm based on measurements in our laboratory (unpublished). Hence, we inserted the pipette no deeper than 8 mm, and well before contact with the cervix to avoid possible stimulation of mechanoreceptors, which can cause alteration to the cycle stages in other rodents. The saline was flushed into the vaginal cavity twice, the solution redrawn into the pipette, the pipette removed and the sample expelled onto a glass histological slides (Menzel-Gläser Superfrost, Thermo-Scientific, Braunschweig, Germany). Samples were dried at 27°C for 5 minutes then sprayed with cytology fixative (Spray Fix, Surgipath Medical Industries, Melbourne, Australia). Smearing occurred consecutively for 18 days. Cycle stages were distinguished based on the dominant cell type(s) present in the smear. If the smear appeared to be in a transitional phase, ie, between 2 consecutive stages, each of the stages was designated a time of 0.5 days to the total length. Otherwise, when a smear type was observed, it was designated a period of 1 day to the total length for that stage. Of the n = 18 females subjected to daily lavaging, 4 were omitted from cytological analysis due to non-cycling. All cytological data are based on data from n = 14 females.

For technical comparison, F ₁ (C57BL/6J X CBA/J) hybrid mice (n = 5) sourced from the Monash Animal Research Platform weighing 17–20 g (40–60 days old) were smeared daily for 12 consecutive days. All aspects of animal handling, vaginal sample collection, and smearing technique were the same as described for the spiny mouse above, except use of a protective hand towel was not necessary.

Cytology staining

Slides were stained with hematoxylin-eosin (Harris hematoxylin and 1% aqueous eosin; Amber Scientific, Midvale, Australia). Briefly, slides were rehydrated in running tap water for 30 seconds –1 minute, prior to hematoxylin staining for 5–6 minutes. Slides were then washed 3 times in tap water to remove excess stain and differentiated in 5% acid ethanol. Following rinsing, slides were submerged repeatedly in a 5% ammonia solution to develop blue coloration. Slides were counterstained with eosin for 3 minutes. Slides were progressively dehydrated in graded ethanol solutions and underwent 3 successive changes of clearing xylene for 2 minutes each. Each slide was cover-slipped with DPX mounting medium.

Postmortem analysis and tissue collection

Female spiny mice (n = 20) were killed humanely at each distinct stage of the menstrual cycle (n = 4/cycle stage). Each female was heavily sedated using isoflurane before a cardiac puncture was performed for blood collection, followed immediately by cervical dislocation. Whole blood (0.3–1.2 mL per animal) was collected in a heparin-lined tube and plasma obtained after centrifugation (3000 RPM at 4°C for 10 minutes), which was then stored at –20°C for later analysis. The whole, intact uterus with both ovaries and the cervix attached was trimmed of fat and removed. Ovaries were then separated, and the uterus weighed (wet weight) before fixation in 10% buffered formalin for 48 hours, followed by immersion in 70% ethanol for 24–72 hours. Samples were processed to paraffin wax using a Leica ASP-300 processer and embedded in Paraplast paraffin medium; then 5 μm-thick sections were obtained, adhered to slides, and baked at 60°C for 20–30 minutes.

Histological staining

Samples were dewaxed through successive xylene changes, cleared in graded ethanol, and rehydrated in tap water. To visualize reproductive tissues, slides were stained with Mallory’s trichrome. Briefly, sections underwent a secondary fixation in Bouin’s solution at 60°C for 60 minutes prior to dewaxing. Following this, tissues were stained with acid fuchsin 1 g/100 mL dH ₂ O (distilled water) for 2 minutes, rinsed thoroughly in distilled water, and stained with phosphomolybdic acid 1 g/100 mL dH ₂ O for 2 minutes. Slides were rinsed before staining with orange G 2 g, methyl blue 0.5 g, and oxalic acid 2 g/100 mL dH ₂ O for 15 minutes. Following thorough rinsing, tissues were dehydrated and differentiated as for hematoxylin-eosin above. Red staining was indicative of nuclei and muscle, blue staining indicated collagen, and orange staining indicated anucleate cells (eg, erythrocytes).

Progesterone assay

Plasma progesterone (P4) concentration was measured using a commercially available mouse/rat enzyme-linked immunosorbent assay kit (ALPCO, no. 55-PROMS-E01). All samples (n = 5 females per cycle stage) were measured in duplicate and the median value reported. The validity of the assay for spiny mouse plasma was tested by spike and recovery and linearity of dilution procedures following the manufacturer’s protocols. Selected samples from individual animals were spiked with 0, low (10 ng/mL), medium (25 ng/mL), or high (50 ng/mL) analyte (progesterone powder, Sigma-Aldrich, Castle-Hill, Australia) and 1 sample diluted by a factor of 4, 8, and 16, and the expected vs observed recovery measured.

Immunohistochemistry

Fixed tissues were subjected to immunohistochemistry to detect presence of decidualization using prolactin as biomarker. Samples were dewaxed through multiple changes of histolene, and rehydrated through decreasing concentrations of ethanol until submerged in running tap water for 1 minutes. All washing steps occurred in TBS-Tween 20 (0.1%) buffer solution. Slides were first washed for 3 × 5 minutes before exogenous peroxidase activity was blocked by applying 1% H ₂ O ₂ solution for 20 minutes. Slides were washed 3 times in buffer and serum block was applied for 30 minutes at room temperature (X090930-2, Dako, Botany, Australia). Slides were incubated in primary polyclonal rabbit antihuman prolactin (A0569, Dako) at 1:400 dilution in 10% normal goat serum at 4°C overnight or 10% normal goat serum as a negative control. Slides were thrice washed and incubated in secondary biotinylated goat antirabbit IgG (Vector Laboratories, CA) at 1:200 dilution for 60 minutes at room temperature. Following washing, slides were incubated in avidin-biotin complex at room temperature for 60 minutes, then washed in buffer solution. 3,3’-Diaminobenzidine was then applied for 6 minutes for visualization. Slides were washed in buffer 3 times and washed again in running tap water before dehydration in increasing graded ethanol. Slides were cleaned and coverslipped as in the hematoxylin-eosin protocol outlined above. Uterine tissue from an early gestation (7 days postconception) pregnant spiny mouse was used as a positive control for prolactin staining (not shown).

Image acquisition

Images of whole organs were captured using an iPhone 4 (iPhone 4, Apple, Cupertino, CA). All histological images were captured using a Leitz Diaplan imaging bright-field microscope (Diaplan, Leitz, Baden-Württemberg, Germany), with light settings kept constant and analyzed with the programs Axio-Vision version 4.6.3 (Zeiss, Oberkochen, Germany) and Image Pro-Plus version 6 (Media Cybernetics, Tokyo, Japan). Images of the vaginal cytology of both the spiny mouse and F ₁ mouse were taken at ×200 magnification. Histological images of the cervix and ovaries of the spiny mouse were taken at ×40 magnification, and the sections of the uterine horns taken at ×100 and ×200 magnification. Using Axio-Vision, the thickness of the endometrium and myometrium were measured, as was the diameter of the uterine lumen (n = 4 animals per stage receiving 3 random recordings per uterus to give n = 12 measurements per stage; μM), across all stages of the reproductive cycle in the spiny mouse. The degree of immunopositive staining was determined using Image Pro-Plus, as described by Atik in 2014. Calibration of the light and dark fields was implemented by the use of a control image containing both incident light and infinite optical density; brought about by use of a scalpel blade placed on the stage to partially obscure the light. All images were captured in 1 session, with all program settings and light exposure kept exact for each section. Images were converted to grayscale and the mean optical density of positive prolactin-stained areas of the endometrium and myometrium was measured. Mean values were calculated for each section, with 3 fields of view at ×200 magnification per structure of interest (endometrium or myometrium) per animal. Regions containing background staining were measured for each image and subtracted from the mean optical density of each sample to adjust for nonspecific staining. The average optical density was calculated for each group. The investigators conducting measurements of the reproductive tract and optical density were blinded to the treatment groups at the time of analysis.

Image alterations

After completion of analysis, images were subjected to minor aesthetic alterations using Adobe Photoshop CC (Adobe Systems, San Jose, CA). Images were sharpened, and backgrounds brightened if required. Due to the variability in staining, color balancing was performed on some histological images to match specific sections of tissues for easier comparison.

Statistical analysis

All statistical analyses were conducted using SPSS (Version 22) (IBM, New York, NY) and GraphPad Prism (Version 6.01) (Graphpad Software, La Jolla, CA). Normality of the data was tested before statistical analysis. Uterine weight, endometrial and myometrial thickness, uterine lumen diameter, and plasma progesterone concentrations (n = 5 samples per stage) are displayed as the median values of replicates (with 25 and 75 percentile values) and significant differences were calculated using Kruskal-Wallis. Optical density and menstrual cycle length are reported as the mean ± SEM and significant differences (corresponding to a P value of < .05) were calculated using a 1-way analysis of variance. Tukey multiple comparisons test was used for comparing parameters between stages of the reproductive cycle during post-hoc analysis.

Animal care

All experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and all experiments approved by the Monash University/Monash Medical Centre Animal Ethics Committee. These animals were sourced from our own research colony, where temperature is maintained at 25–27°C, humidity 30–40%, with a 12-hour light-dark cycle (lights on 7:00 am ). Virgin female spiny mice (n = 18) aged 12–16 weeks, were housed in groups of 5–6 per cage. Cages were lined in wooden shavings and enrichment provided in forms of cardboard tunnels and climbing apparatuses. Food (rat and mouse cubes, Specialty Feeds, Glen Forrest, WA) and water were provided ad libitum and supplemental fresh vegetables (carrots and celery, up to 50 g per cage) were provided weekly. Animals were checked at least twice daily and cages were cleaned weekly. Female weights were monitored daily throughout the experiment.

Vaginal lavage

Vaginal lavages were performed daily (n = 18 females) between 12:30 pm and 2:30 pm using sterile saline solution (0.9% wt/vol), and the time of sampling recorded each day. Female spiny mice were restrained in the supine position with a small hand towel by gently scruffing behind the neck and shoulders (a hand towel is necessary due to the fragility of spiny mouse connective tissues and the propensity for skin to easily tear). The vulva was then lubricated with nonscented, water-based lubricant (Lifestyles, Ansell, Melbourne, Australia). A 1-mL plastic transfer pipette was used to draw up ∼50 μL of saline, before gentle insertion into the vaginal canal. The length of the spiny mouse vaginal canal is 16.3 ± 1.1 mm based on measurements in our laboratory (unpublished). Hence, we inserted the pipette no deeper than 8 mm, and well before contact with the cervix to avoid possible stimulation of mechanoreceptors, which can cause alteration to the cycle stages in other rodents. The saline was flushed into the vaginal cavity twice, the solution redrawn into the pipette, the pipette removed and the sample expelled onto a glass histological slides (Menzel-Gläser Superfrost, Thermo-Scientific, Braunschweig, Germany). Samples were dried at 27°C for 5 minutes then sprayed with cytology fixative (Spray Fix, Surgipath Medical Industries, Melbourne, Australia). Smearing occurred consecutively for 18 days. Cycle stages were distinguished based on the dominant cell type(s) present in the smear. If the smear appeared to be in a transitional phase, ie, between 2 consecutive stages, each of the stages was designated a time of 0.5 days to the total length. Otherwise, when a smear type was observed, it was designated a period of 1 day to the total length for that stage. Of the n = 18 females subjected to daily lavaging, 4 were omitted from cytological analysis due to non-cycling. All cytological data are based on data from n = 14 females.

For technical comparison, F ₁ (C57BL/6J X CBA/J) hybrid mice (n = 5) sourced from the Monash Animal Research Platform weighing 17–20 g (40–60 days old) were smeared daily for 12 consecutive days. All aspects of animal handling, vaginal sample collection, and smearing technique were the same as described for the spiny mouse above, except use of a protective hand towel was not necessary.

Cytology staining

Slides were stained with hematoxylin-eosin (Harris hematoxylin and 1% aqueous eosin; Amber Scientific, Midvale, Australia). Briefly, slides were rehydrated in running tap water for 30 seconds –1 minute, prior to hematoxylin staining for 5–6 minutes. Slides were then washed 3 times in tap water to remove excess stain and differentiated in 5% acid ethanol. Following rinsing, slides were submerged repeatedly in a 5% ammonia solution to develop blue coloration. Slides were counterstained with eosin for 3 minutes. Slides were progressively dehydrated in graded ethanol solutions and underwent 3 successive changes of clearing xylene for 2 minutes each. Each slide was cover-slipped with DPX mounting medium.

Postmortem analysis and tissue collection

Female spiny mice (n = 20) were killed humanely at each distinct stage of the menstrual cycle (n = 4/cycle stage). Each female was heavily sedated using isoflurane before a cardiac puncture was performed for blood collection, followed immediately by cervical dislocation. Whole blood (0.3–1.2 mL per animal) was collected in a heparin-lined tube and plasma obtained after centrifugation (3000 RPM at 4°C for 10 minutes), which was then stored at –20°C for later analysis. The whole, intact uterus with both ovaries and the cervix attached was trimmed of fat and removed. Ovaries were then separated, and the uterus weighed (wet weight) before fixation in 10% buffered formalin for 48 hours, followed by immersion in 70% ethanol for 24–72 hours. Samples were processed to paraffin wax using a Leica ASP-300 processer and embedded in Paraplast paraffin medium; then 5 μm-thick sections were obtained, adhered to slides, and baked at 60°C for 20–30 minutes.

Histological staining

Samples were dewaxed through successive xylene changes, cleared in graded ethanol, and rehydrated in tap water. To visualize reproductive tissues, slides were stained with Mallory’s trichrome. Briefly, sections underwent a secondary fixation in Bouin’s solution at 60°C for 60 minutes prior to dewaxing. Following this, tissues were stained with acid fuchsin 1 g/100 mL dH ₂ O (distilled water) for 2 minutes, rinsed thoroughly in distilled water, and stained with phosphomolybdic acid 1 g/100 mL dH ₂ O for 2 minutes. Slides were rinsed before staining with orange G 2 g, methyl blue 0.5 g, and oxalic acid 2 g/100 mL dH ₂ O for 15 minutes. Following thorough rinsing, tissues were dehydrated and differentiated as for hematoxylin-eosin above. Red staining was indicative of nuclei and muscle, blue staining indicated collagen, and orange staining indicated anucleate cells (eg, erythrocytes).

Progesterone assay

Plasma progesterone (P4) concentration was measured using a commercially available mouse/rat enzyme-linked immunosorbent assay kit (ALPCO, no. 55-PROMS-E01). All samples (n = 5 females per cycle stage) were measured in duplicate and the median value reported. The validity of the assay for spiny mouse plasma was tested by spike and recovery and linearity of dilution procedures following the manufacturer’s protocols. Selected samples from individual animals were spiked with 0, low (10 ng/mL), medium (25 ng/mL), or high (50 ng/mL) analyte (progesterone powder, Sigma-Aldrich, Castle-Hill, Australia) and 1 sample diluted by a factor of 4, 8, and 16, and the expected vs observed recovery measured.

Immunohistochemistry

Fixed tissues were subjected to immunohistochemistry to detect presence of decidualization using prolactin as biomarker. Samples were dewaxed through multiple changes of histolene, and rehydrated through decreasing concentrations of ethanol until submerged in running tap water for 1 minutes. All washing steps occurred in TBS-Tween 20 (0.1%) buffer solution. Slides were first washed for 3 × 5 minutes before exogenous peroxidase activity was blocked by applying 1% H ₂ O ₂ solution for 20 minutes. Slides were washed 3 times in buffer and serum block was applied for 30 minutes at room temperature (X090930-2, Dako, Botany, Australia). Slides were incubated in primary polyclonal rabbit antihuman prolactin (A0569, Dako) at 1:400 dilution in 10% normal goat serum at 4°C overnight or 10% normal goat serum as a negative control. Slides were thrice washed and incubated in secondary biotinylated goat antirabbit IgG (Vector Laboratories, CA) at 1:200 dilution for 60 minutes at room temperature. Following washing, slides were incubated in avidin-biotin complex at room temperature for 60 minutes, then washed in buffer solution. 3,3’-Diaminobenzidine was then applied for 6 minutes for visualization. Slides were washed in buffer 3 times and washed again in running tap water before dehydration in increasing graded ethanol. Slides were cleaned and coverslipped as in the hematoxylin-eosin protocol outlined above. Uterine tissue from an early gestation (7 days postconception) pregnant spiny mouse was used as a positive control for prolactin staining (not shown).

Image acquisition

Images of whole organs were captured using an iPhone 4 (iPhone 4, Apple, Cupertino, CA). All histological images were captured using a Leitz Diaplan imaging bright-field microscope (Diaplan, Leitz, Baden-Württemberg, Germany), with light settings kept constant and analyzed with the programs Axio-Vision version 4.6.3 (Zeiss, Oberkochen, Germany) and Image Pro-Plus version 6 (Media Cybernetics, Tokyo, Japan). Images of the vaginal cytology of both the spiny mouse and F ₁ mouse were taken at ×200 magnification. Histological images of the cervix and ovaries of the spiny mouse were taken at ×40 magnification, and the sections of the uterine horns taken at ×100 and ×200 magnification. Using Axio-Vision, the thickness of the endometrium and myometrium were measured, as was the diameter of the uterine lumen (n = 4 animals per stage receiving 3 random recordings per uterus to give n = 12 measurements per stage; μM), across all stages of the reproductive cycle in the spiny mouse. The degree of immunopositive staining was determined using Image Pro-Plus, as described by Atik in 2014. Calibration of the light and dark fields was implemented by the use of a control image containing both incident light and infinite optical density; brought about by use of a scalpel blade placed on the stage to partially obscure the light. All images were captured in 1 session, with all program settings and light exposure kept exact for each section. Images were converted to grayscale and the mean optical density of positive prolactin-stained areas of the endometrium and myometrium was measured. Mean values were calculated for each section, with 3 fields of view at ×200 magnification per structure of interest (endometrium or myometrium) per animal. Regions containing background staining were measured for each image and subtracted from the mean optical density of each sample to adjust for nonspecific staining. The average optical density was calculated for each group. The investigators conducting measurements of the reproductive tract and optical density were blinded to the treatment groups at the time of analysis.

Image alterations

After completion of analysis, images were subjected to minor aesthetic alterations using Adobe Photoshop CC (Adobe Systems, San Jose, CA). Images were sharpened, and backgrounds brightened if required. Due to the variability in staining, color balancing was performed on some histological images to match specific sections of tissues for easier comparison.

Statistical analysis

All statistical analyses were conducted using SPSS (Version 22) (IBM, New York, NY) and GraphPad Prism (Version 6.01) (Graphpad Software, La Jolla, CA). Normality of the data was tested before statistical analysis. Uterine weight, endometrial and myometrial thickness, uterine lumen diameter, and plasma progesterone concentrations (n = 5 samples per stage) are displayed as the median values of replicates (with 25 and 75 percentile values) and significant differences were calculated using Kruskal-Wallis. Optical density and menstrual cycle length are reported as the mean ± SEM and significant differences (corresponding to a P value of < .05) were calculated using a 1-way analysis of variance. Tukey multiple comparisons test was used for comparing parameters between stages of the reproductive cycle during post-hoc analysis.