In rats, female sexual behavior is regulated by a well defined limbic-hypothalamic circuit that integrates sensory and hormonal information. Estradiol activation of this circuit results in -opioid receptor (MOR) internalization in the medial preoptic nucleus, an important step for full expression of sexual receptivity. Estradiol acts through both membrane and intracellular receptors to influence neuronal activity and behavior, yet the mechanism(s) and physiological significance of estradiol-mediated membrane responses in vivo have remained elusive. Recent in vitro evidence found that stimulation of membrane-associated estrogen receptor-␣ (ER␣) led to activation of metabotropic glutamate receptor 1a (mGluR1a). Furthermore, mGluR1a signaling was responsible for the observed downstream effects of estradiol. Here we present data that show that ER␣ and mGluR1a directly interact to mediate a rapid estradiol-induced activation of MOR in the medial preoptic nucleus, leading to female sexual receptivity. In addition, blockade of mGluR1a in the arcuate nucleus of the hypothalamus resulted in a significant attenuation of estradiol-induced MOR internalization, leading to diminished female sexual behavior. These results link membrane-initiated estradiol actions to neural events modulating behavior, demonstrating the physiological importance of ER␣-to-mGluR1a signaling.

The goal of this morphometric study was to obtain quantitative information on the seminiferous tubules of Sprague-Dawley rats, including changes seen at various stages of the cycle of the seminiferous epithelium. Tissue from perfusion-fixed testes was embedded in Epon-Araldite; and sections were subjected to morphometric measurements at the light microscopic level, using point counting for volume densities and the Floderus equation for numerical densities. Changes occur in the diameter of the seminiferous tubule, as well as in the volume of the seminiferous epithelium and tubule lumen, from stage to stage during the cycle. A significant constriction of the seminiferous tubule accompanies spermiation. The volume of the seminiferous epithelium per unit length of the tubule begins to increase after stage XIV, and peaks at stage V of the next cycle. The tubule lumen increases dramatically from stages V to VII, at the expense of the epithelium. The number of Sertoli cells is constant per unit length of the seminiferous tubule at all stages of the cycle. This is also true for primary spermatocytes of various developmental phases and for round spermatids from step 1 through step 10 of spermiogenesis. The average number of younger (preleptotene, leptotene, zytgotene) primary spermatocytes per Sertoli cell is 2.34 +/- 0.082 (SEM), the number of older (pachytene, diplotene) primary spermatocytes per Sertoli cell is 2.37 +/- 0.064, and the ratio of step 1-10 spermatids to Sertoli cells is 7.89 +/- 0.27. By studying tangential views of serially sectioned seminiferous tubules at stage V, it is shown that the number of step-17 spermatids associated with each Sertoli cell averages 8.35 +/- 0.128, although the counts ranged from 6 to 11. The only appreciable occurrence of cell death after the last spermatogonial mitosis appears to be a 15% loss during the first meiotic division. From our morphometric results, corrected for volume changes during preparation for microscopy, there are 15.7 million (+/- 0.99 million) Sertoli cells per gram of fresh rat testis. The length of seminiferous tubule per gram of testis is estimated to be 12.4 +/- 0.56 meters, and the tubule surface area per gram testis is 119.7 +/- 2.57 cm2. The daily production of mature spermatids is 9.61 million (+/- 0.615 million) per gram of testis.

Leydig cells are thought to be the source of most, if not all, the testosterone produced by the testis . The goal of this study was to obtain quantitative information about rat Leydig cells and their organelles that might be correlated with pertinent physiological and biochemical data available either now or in the future . Morphometric analysis of Leydig cells in mature normal rats was carried out on tissue fixed by perfusion with buffered glutaraldehyde, and embedded in glycol methacrylate for light microscopy and in Epon for electron microscopy .In a whole testis, 82.4% of the volume was occupied by seminiferous tubules, 15 .7% by the interstitial tissue, and 1.9% by the capsule. Leydig cells constituted 2.7% of testicular volume . Each cubic centimeter (-1 g) of rat testis contained about 22 million Leydig cells. An average Leydig cell had a volume of 1,210 Am3 and its plasma membrane had a surface area of 1,520 Am'.The smooth endoplasmic reticulum (SER), the most prominent organelle in Leydig cells and a major site of steroidogenic enzymes, had a surface area of 10,500 ym2/cell, which is 6.9 times that of the plasma membrane and is 60% of the total membrane area of the cell . The total surface area of Leydig SER per cubic centimeter of testis tissue is 2,300 cm' or 0.23 mz . There were 3 .0 mg of Leydig mitochondria in 1 g of testis tissue . The average Leydig cell contained -622 mitochondria, measuring on the average 0.35 Am in diameter and 2.40 Am in length. The mitochondrial inner membrane (including cristae), another important site of steroidogenic enzymes, had a surface area of 2,920 Am'/cell, which is 1 .9 times that of the plasma membrane . There were 644 cm' ofinner mitochondrial membrane/cm' of testis tissue .These morphometric results can be correlated with published data on the rate of testosterone secretion to show that an average Leydig cell secretes -0.44 pg of testosterone/d or 10,600 molecules of testosterone/s . The rate of testosterone production by each square centimeter of SER is 4.2 ng/d or 101 million molecules/ s; the corresponding rate for each square centimeter of mitochondrial inner membrane is 15 ng testosterone/d or 362 million molecules/s. 340J . CELL BIOLOGY

A simple method has been developed that allows frozen thin sections of fresh-frozen tissue to be cut on a virtually unmodified ultramicrotome kept at room temperature. A bowl-shaped Dewar flask with a knifeholder in its depths replaces the stage of the microtome; a bar extends down into the bowl from the microtome's cutting arm and bears the frozen tissue near its lower end. When the microtome is operated, the tissue passes a glass or diamond knife in the depths of the bowl as in normal cutting. The cutting temperature is maintained by flushing the bowl with cold nitrogen gas, and can be set anywhere from about -160°C up to about -30°C. The microtome is set for a cutting thickness of 540–1000 A. Sections are picked up from the dry knife edge, and are placed on membrane-coated grids, flattened with the polished end of a copper rod, and either dried in nitrogen gas or freeze-dried. Throughout the entire process the tissue is kept cold and does not come in contact with any solvent. The morphology seen in frozen thin sections of rat pancreas and liver generally resembles that in conventional preparations, although freezing damage and low contrast limit the detail that can be discerned. Among unusual findings is a frequent abundance of mitochondrial granules in material prepared by this method.

Rapid membrane-mediated estradiol signaling regulating sexual receptivity requires the interaction of the estrogen receptor (ER)-alpha and the metabotropic glutamate receptor 1a (mGluR1a). A cell signaling antibody microarray revealed that estradiol activated 42 proteins in the arcuate nucleus of the hypothalamus (ARH). To begin an analysis of various signaling pathways, protein kinase A and protein kinase C (PKC)-theta, whose signaling pathways have been implicated in the estradiol regulation of sexual receptivity, were examined. In the ARH sample, the increase in phospho-protein kinase A could not be confirmed by Western blotting, in either cytosolic or membrane fractions. However, the increase in phosphorylated PKCtheta seen with the pathway array was verified by Western blotting. To study whether rapid estradiol activation of PKC regulates the ARH-medial preoptic nucleus pathway regulating lordosis, mu-opioid receptor (MOR) internalization and lordosis reflex were tested. Blocking PKC in ARH with 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]3-(1H-indol-3-yl) maleimide significantly attenuated estradiol-induced MOR internalization. Furthermore, disruption of PKC signaling within the ARH at the time of estradiol treatment significantly diminished the lordosis reflex. Moreover, blocking PKC prevented MOR internalization when the circuit was activated by the mGluR1a agonist, (RS)-3,5-dihydroxyphenylglycine. Activation of PKC with phorbol 12, 13-dibutyrate induced MOR internalization, indicating that PKC was a critical step for membrane ERalpha-initiated mGluR1a-mediated cell signaling and phorbol 12, 13-dibutyrate significantly facilitated the lordosis reflex. Together these findings indicate that rapid membrane ERalpha-mGluR1a interactions activate PKCtheta cell signaling, which regulates female sexual receptivity.