Novel-Tissue Specifc Sex specific through estrous cycle

Serum sex steroid levels fluctuate throughout the reproductive cycle.
Objective: Understand how sex steroid tissue content mimics circulating levels and its importance for hormonal therapy development.
Method: Used ultrasensitive liquid chromatography–mass spectrometry (LC/MS) to measure fluctuations of sex steroids (17β-estradiol [E2], testosterone, androstenedione, progesterone) in serum and 15 tissues throughout the murine estrous cycle (proestrus, estrus, diestrus I) and in ovariectomized (OVX) mice.
Findings:
- Highest content of E2, testosterone, and androstenedione generally during proestrus, while progesterone was lowest.
- Serum steroid levels were typically lower than tissue levels when normalized to tissue weight.
- OVX mice showed depletion of all steroids except in adrenal glands, which became the primary site for peripheral E2 production.
Implication: Data serves as groundwork for developing tissue-specific hormonal therapies.

Importance of Sex Steroids:
- Essential for several physiological processes, such as metabolism, sexual differentiation, reproduction, skeletal, and immune function.
- Both sexes possess estrogens, androgens, and progesterone, but differ in absolute levels: males have higher androgens, while females have more estrogens and progesterone.
Fluctuations in Serum Levels:
- Female hormone levels vary during menstrual and estrous cycles but previously limited by sensitivity in methodologies.
- Understanding tissue fluctuations critical to developing targeted hormone therapies.
Research Gap:
- Previous studies lacked clarity on how tissue sex steroid content mimics circulating levels effectively across the reproductive cycle.

To examine tissue sex steroid levels during the murine estrous cycle and in OVX mice, and to establish a basis for hormone treatments achieving physiological levels.

Mouse Model: C57BL/6 background, housed 3-5/cage, 22 °C, 12:12 light-dark cycle (07:00-19:00 hours).
Age: 12-16 weeks at experiment start.
OVX Procedure: Bilateral ovariectomy performed at approximately 12 weeks of age (n = 15). Mice sacrificed for tissue collection 3 weeks post-surgery.
Diet: Purified, open-source AIN-76A diet provided (Bio-Serv, Frenchtown, NJ).

Purpose: Monitor estrous cycles through daily vaginal lavages (07:30 – 9:30 hours) for 2 consecutive cycles.
Technique:
- 10 µL sterile-filtered phosphate-buffered saline applied and collected for cytological examination.
- Stains and imaging performed after drying.
Cycle Staging:
- Proestrus: Predominantly nucleated epithelial cells with smooth edges.
- Estrus: Presence of cornified epithelial cells.
- Diestrus I: Predominantly leukocytes.

Timing: Sacrifice between 09:00 – 10:30 hours post-cytology.
Procedure: Mice euthanized via isoflurane inhalation; blood collected and processed.
Organs Collected: Ovaries (unless OVX), adrenal glands, uterus, various adipose tissues, pancreas, skeletal muscle, liver, kidneys, spleen, colon, lungs, heart, and flash frozen.

Sources: Blood serum and tissue weighed for extraction. Internal standards (Sigma Aldrich) added for quantitative analysis.
Extraction Technique:
- Blood serum extracted using methyl tert-butyl ether (MTBE); tissue homogenized in acetonitrile, centrifuged, and subjected to liquid-liquid extraction.
- Derivatized samples analyzed mass spectrometrically.

Equipment Used: Q Exactive HF-X quadrupole-orbitrap mass spectrometer; Waters Xbridge C18 column.
Methods of Analysis:
- E2: Targeted selected ion monitoring.
- Other Steroids (Testosterone, Androstenedione, Progesterone): Parallel reaction monitoring.
Calibration: Calibration curves with certified reference materials determined concentration levels.

Conducted using Prism 9 (GraphPad Software).
Analysis included 1-way ANOVA and Newman–Keuls post hoc tests with significance level set at P < .05.

Observation: Uterus most massive during proestrus, followed by estrus, and least in diestrus I. OVX uterus presented lowest weight.

Peak Levels: Detected highest during proestrus across 14/16 tissues. No significant E2 fluctuation in adrenal gland between proestrus and diestrus I.
Trends: Tissue E2 levels generally followed circulation trends.
Comparative Metrics:
- Ovaries, uterus, and adrenal glands had the highest tissue E2 levels, more significant variances noted in visceral and subcutaneous adipose compared to brown adipose.

Round Analysis: Higher levels during proestrus in ovaries, serum, and select adipose tissues (6/16 tissues) compared to all stages.
OVX Effect: OVX mice exhibited a greater testosterone level in adrenal glands than intact mice.

Pattern: Highest during proestrus in 9/16 tissues; OVX mice displayed significantly lower serum levels.
Summary of Sites: Primary sections producing high levels were ovaries and adrenal glands; adipose tissue depots supported largest sites of androstenedione content.

Observation: Proestrus and OVX groups presented lower progesterone levels relative to estrus and diestrus groups across various tissues.
Tissue Comparison: Highest in ovaries and adrenal glands; lower in other tissues such as liver and spleen.

Methodology Importance: Emphasizes the application of an ultrasensitive LC-MS method to advance sex steroid measurement in murine studies.
Gender Comparisons: Discrepancies between human and mouse models concerning hormonal production and metabolism, highlighting that systemic treatment effects and bioavailability need further understanding.
Clinical Implications: Data lays foundation for optimizing hormone replacement therapies targeting physiological levels.