Electron Microscopy and Histochemical Correlation of Human Anterior Pituitary Cells Carlos Paiz, MD and Gordon R. Hennigar, MD THE PARS ANrERIOR of human adenohypophysis has been studied extensively by histochemical and tinctorial methods."-8 Multiple nomenclatures have been developed to describe the various cell types, but the ultimate goal is to adopt a terminology based solely on function. Electron microscopy has served to clarify in part the fine structure of the adenohypophysis, and its contribution to date has been the attempted correlation of granular size and shape with specific hormonal secretion.9-" Similar studies have been made in animal species other than man.12-'7 In several human cell types, the granules are so similar that it is fre- quently difficult if not impossible to distinguish the cells on morphologic grounds alone. The use of thick-thin section correlation for light and electron microscopy can, in part, clarify this problem, since histochem- ical properties at the light level may be correlated with fine structural differences within the same cell at the electron microscopic level. Such correlations have served three purposes: (1) We have been able to relate certain serous, mucoid, and seemingly chromophobe cells of light microscopy with the corresponding electron microscopic equiv- alents. (2) In so doing, we have demonstrated that cells having the same or similar granule morphology with electron microscopy are strik- ingly different with light microscopy histochemistry. (3) The thick-thin section method of comparison has provided us with the opportunity to relate fine structural differences within pituitary cells with the corre- sponding variable dye binding seen in a single cell with light micros- copy. Materials and Methods Adenohypophyses were obtained from 11 cases of necropsy and in 1 instance following surgical hypophysectomy (Table 1). The adenohypophysis obtained at surgery was received in small fragments so From the Departments of Pathology of the State University of New York Downstate Medical Center, Brooklyn, NY, and Veterans Administration Hospital, Medical University of South Carolina, Charleston, SC. Supported by NIH Grants CA06081 and AM10956. Accepted for publication Oct 14, 1969. Address for reprint requests: Dr. Gordon R. Hennigar, Department of Pathology, Med- ical University of South Carolina, 80 Barre St, Charleston, SC 29403. 43 44 PAIZ AND HENNIGAR American Journal of Pathology Table 1. Survey of Cases of Human Adenohypophysis Interval between death No. Age Sex Race and fixation Diagnosis Cause of death 1 40 M W 6hr 50min Diabetes mellitus Heart failure 2 52 M W 10hr 00min Diabetes mellitus Acute myocardial infarction 3 87 F W 3hr 45min Bronchopneumonia Bronchopneumonia 4 51 M PR lhr 00min Laennec's cirrhosis Acute abdominal hemorrhage 5 37 M N 8hr 15min Alcoholism Bronchopneumonia 6 33 F N 6hr 00min Pregnancy Abortion; Uterine rupture 7 60 F W 4hr 30min Obesity Bronchopneumonia 8 53 M W 2hr 10min Laennec's cirrhosis Postoperative shock 9 29 F N 32hr 00min Suicide 10 35 M N 9hr 15min Homicide 11 22 M W 16hr 00min Suicide 12 62 F W Ohr 10min Ca of breast Surgical specimen; alive that it was impossible to determine the anatomic site of origin of the cells. On the other hand, necropsy specimens could be oriented as to anatomic site. The glands were first cut in their greatest horizontal diameter. A 1-mm slice of tissue following the original plane of section was then made and the tissue was placed on a sheet of dental wax and covered with several drops of glutaraldehyde. Tissue excised for electron microscopy was obtained from three different zonal sources labeled A, B, and C (Text-fig 1). Five 1-cu mm tissue blocks were obtained from each zone of the horizontal sections and fixed 1 hr at 6°C in Sabatini's glutaraldehyde.18 The tissue was then washed in three changes of phosphate buffer (30 min each) and then postosmicated (1 hr) in 1% osmium tetroxide in Palade's phosphate buffer (pH 7.4) 19 containing sucrose. The blocks were rapidly dehydrated through graded alcohol solutions and embedded in Epon.20 Thin sections were cut with a Huxley ultramicrotome and stained with either lead hydroxide or uranyl acetate.21 The adjacent thick section was treated with a solution of 3% NaOH in 80% ethyl alcohol for 5 min. This solution dissolved the embedding media and facilitated the staining of the thick section by PAS-Orange G sequence. Magnifications in the electron microscope were determined against a carbon grating replica calibration standard (No. 115) with 28,800 lines/linear in. (Ern- est F. Fullam, Inc., Schenectady, NY). The time elapsed between the death of the individual and fixation of the pitui- tary varied from 30 min to 32 hr. The surgical specimen was fixed within 10 min of its removal. This relatively wide range provided us with some preliminary basis for the evaluation of pituitary cell preservation following various postmortem in- tervals. Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 45 April 1970 ZONAL SOURCE ANT. OF SAMPLE TEXT-FIG 1. Ant = anterior, P = posterior, pars nervosa, A = serous wing, B = mucoid wedge, C = postenror mucoid wedge. Tissue excised for electron microscopy ob- tained from three different zonal sources labelled A, B, and C. Zone A corresponds to lateral wings of adenohypophysis, and is heavily populated by "serous or acidophil cells." Zone B consisted of anteromedial portion of gland which has a predominantly mucoid cell population. Zone C was the area equivalent to the pars intermedia in other than human animal species. One surgical specimen (Case 12) and one necropsy specimen (Case 6) were correlated by the adjacent thick-thin section method. The same cell field in elec- tron micrographs was compared to the adjacent section stained by PAS-Orange G.22,23 Results Although postmortem autolysis is fairly rapid, necropsy material may in part be suitable for limited electron microscopic investigation. How- ever, in cases of sudden death, glands obtained up to 8 hr postmortem revealed better preservation than those obtained at a shorter postmortem interval from people with severe illnesses. It was observed that shock and anoxia play an important role in the acceleration of cytolysis. The McManus PAS procedure22 followed by an Orange G counter- stain enables differentiation of pituitary cells into 3 large groups: the orangeophils or serous cells; the PAS-positive reacting group or mucoid cells; 23 and a large group of colorless cells or chromophobes. Electron microscopically, each group may be subdivided according to the cyto- plasmic characteristics of the different cell types. The serous cells comprise a heterogeneous and large population which predominates in the lateral wings of the pars anterior (Text-fig 1). For practical purposes they are subdivided into cells with large or small secretory granules. The acidophils with large granules are the most common. Their secre- 46 PAIZ AND HENNIGAR American Journal of Pathology tory granules are uniformly dense, varying in size from 250 m,u to 500 mp with a mean diameter of 350 mu (Fig 1 and 4). The smallest granula- tions are round and the largest somewhat irregular, varying in shape from round to oval to piriform. The ergastoplasm is prominent, com- posed of flattened cisternae which are sometimes arranged in parallel rows or whirlpools. In some acidophils with large granules a faint PAS reaction was observed about the centrosphere (Fig 2 and 6). At higher magnification this PAS-positive area (Fig 7) is seen to contain a lamellar and tubular Golgi complex and a few seemingly immature or early gran- ules. The second type of acidophil with smaller granules is less common. The smaller granules within it have a clear area between the granular core and its plasma membrane. The larger granules which measured from 200 to 300 ma are more electron dense and there is no distinct perigranular clear zone (Fig 8). The ergastoplasm is poorly developed and composed of minute vesicles and short tortuous cisternae. The hyaloplasm is abundant, with a moderate amount of free ribosomes and microtubules. The mitochondria are scarce (Fig 5). A section 2 ,M thick usually fails to demonstrate the serous nature of the cell. However, if the section thickness is increased to 4 or 6 u the cell acidophilia is demonstrable, provided sufficient numbers of granules are present. The PAS-positive or mucoid cells are also subdivided into cells charac. terized by large and small granules. The most common form is the cell with small granules. The secretory characteristics are manifested by a very electron-dense area and are ovoid to pear-shaped. The granular dimensions in the maximally granulated cell range from 300 to 800 mp with a mean of 450 m,&. The ergastoplasm is well developed and com- posed of flat cisternae. These cells are seen more commonly topograph- ically positioned in the middle and anterior portion of pars distalis. On occasion it is impossible to differentiate this cell from the serous cell with 350 mu diameter granules since their smaller granulations overlap considerably and the ergastoplasm is similar (Fig 3 and 9). The second, less common type of PAS-positive mucoid cell, which has large granules, is usually found in tissue blocks from the most ante- rior portion of pars distalis. This mucoid cell is readily identified electron microscopically due to the peculiar characteristics of their secretory granules which are packed together during the storage phase. Furthermore, the secretory granules are of variable density with the smaller sizes being more electron dense and the larger less electron Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 47 April 1970 dense. Both types of secretory granules measure from 350 to 1100 mu. Occasionally we have observed cells with low-density granules up to 2000 mu in diameter.