Structural response of the hamster Sertoli cell to hypophysectomy: A correlative morphometric and...

THE ANATOMICAL RECORD 234513-529 (1992)

Structural Response of the Hamster Sertoli Cell to Hypophysectomy : A Correlative Morphometric and Endocrine Study

SUSHMITA GHOSH, ANDRZEJ BARTKE, PATRICIA GRASSO, LEO E. REICHERT, JR., AND LONNIE D. RUSSELL

Laboratory of Structural Biology, Department of Physiology, Southern Illinois University, School of Medicine, Carbondale, Illinois (S.G., A.B., L.D.R.); Department of Biochemistry

and Molecular Biology, The Albany Medical College, Albany, New York (P.G., L.E.R.)

ABSTRACT Reproductively active hamsters were hypophysectomized and examined 6 or 20 days later in a combined morphometric and endocrine study of the Sertoli cell to determine 1) the morphological and endocrine effects of hypophysectomy of both short- and long-term duration, 2) if regression of Sertoli cells after hypophysectomy in a seasonal breeder re- sembles regression due to seasonal changes, and 3) if effects of hypophysectomy in a seasonal breeder are equivalent to the effects of hypophysectomy in a nonseasonal breeder. Six days after hypophysectomy, at a period when germ cell degeneration is first noted, there was a significant decrease in testis weight, interstitial space, tubule diameter and length, volume of seminiferous tubule, and tubular lumen. There were no significant changes in Sertoli cell nuclear and cytoplasmic volume although cell surface area was decreased significantly. Most organelles exhibited no significant change in volume or surface area except for secondary lysosomes which expectedly increased in volume as the result of phagocytosis of germinal cells. Thus at an early time period when functional changes in germ cells and Leydig cells are clearly evident (Russell et al. [19921 Endocrinology), the Sertoli cell shows minimal changes. Twenty days after hypophysectomy, the cell, nuclear and cytoplasmic volumes and surface area of the Sertoli cells, and volumes and surface areas of nearly all organelles were significantly decreased from values measured in normal and in short-term hypophysectomized hamsters. The exceptions were the total volumes of lipid which increased significantly and lysosomes which were similar to normal but significantly lower than short-term hypophysectomized animals. The long-term hypophysectomized hamster Sertoli cell, like that of the short-day hamster (Sinha Hikim et al. [1989bl Endocrinology, 125:1829- 1843) is structurally regressed as a whole rather than exhibiting selective decreases in cellular and subcellular components. The size of the Sertoli cell in pituitary-intact, long- and short-term hypophysectomized animals showed positive and significant correlations with the volumes and surface areas of all its cytoplasmic organelles except the volume of lipid which showed a negative, significant correlation. Comparisons of long-term hypophysectomized hamsters (in long-day light exposure) and short-day exposed animals (Sinha Hikim et al. [1989bl (Endocrinology, 125:1829-1843) suggested that hypophysectomy, in general, resulted in similar, but slightly more severe regressive changes in the testis and germ cell population than those seen during seasonal regression. This was manifest in the Sertoli cell as a significantly lower cell surface area and nuclear volume although the majority of cytoplasmic organelles of the Sertoli cell were not significantly different, nor were plasma follicle stimulating hormone (FSH) and testosterone levels dissimilar. With respect to the Sertoli cell, the structural manifestations of hypophysectomy in the hamster are strikingly similar to those

Received January 6, 1992; accepted March 16, 1992. Address reprint requests to L.D. Russell, Laboratory of Structural

Biology, Department of Physiology, Southern Illinois University, School of Medicine, Carbondale, IL 62901-6512.

0 1992 WILEY-LISS, INC.

514 S. GHOSH ET AL.

detected in the rat (Ghosh et al. 119921 Endocrinology) with one exception that the Sertoli cell surface area in the rat does not decrease significantly in the short term. In correlation tests of morphological parameters and hormone levels in longday, short-term and long-term hypophysectomized hamsters, several parameters were correlated with plasma FSH and testosterone levels. The content, but not the concentration (expressed per basal compartment surface area), of FSH receptors decreased in short-term hypophysectomized animals. Compared to the Leydig cell, the Sertoli cell structure responds slowly to hypophysectomy, but, like the Leydig cell, is markedly changed in the long term.

Key words: Hamster, Hypophysectomy, Photoperiodic, Spermatogenesis, Sertoli cell, Testosterone, Luteinizing hormone, Follicle-stim-

o 1992 Wiley-Liss, Inc.

ulating hormone

Seasonal breeders such as the Syrian (golden) hamster (Mesocricetus uurutus) exhibit an annual cycle of reproduction that is related in nature to changes in the light-dark cycle. Cyclic changes in reproductive capa- bility can be induced in the laboratory by creating artificial light-dark cycles (reviews in Bartke, 1985; Reiter, 1985). After exposing gonadally active male hamsters to a short photoperiod, their testes undergo regression. Returning the animals to a long photoperiod can reverse this condition such that the gonads once again become functional (Matt and Stetson, 1979; Nelson and Zucker, 1987). Accompanying the short photoperiod-induced atrophy of the testis is a decrease in serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and testosterone (Berndtson and Desjardins, 1974; Turek et al., 1975; Bex et al., 1978). There is also a decrease in the number of receptors for FSH, LH, and PRL (Bartke, 1985; Bartke et al., 1987). Similar changes can be brought about by blinding (Reiter, 1968; Desjardins et al., 1971; Gravis, 1977; Gravis and Weaker, 1977). Over the years there have been several studies describ- ing the enocrine changes during the photoperiod- induced reproductive cycle. Changes in the testis have been studied extensively in seasonal breeders, exem- plified by model species such as the hamster (Gaston and Menaker, 1967; Desjardins et ' al., 1971; Reiter, 1980; Hardy et al., 1987; Johnson et al., 1987; Sinha Hikim et al., 1988a,b).

The Leydig cell and Sertoli cell are key cells in the testis that are known to respond to endocrine-induced photoperiodic changes. In short-day animals, these cell types show marked structural changes (Sinha Hikim et al., 1988a) that have been quantitated by stereological techniques to show that the cells and virtually all of their organelles respond to short days by decreases in volume and surface area (Sinha Hikim et al., 1988b, 1989a,b). From these studies, it has been proposed that Leydig cell and Sertoli cell components of the inactive testis of the short-day animal are not selectively shut down, but shut down as a whole. Moreover, there is a high correlation between vitually all cell and organelle parameter decreases and pertinent hormonal parameters.

Hypophysectomy is the classical method to experi- mentally deprive the testis of pituitary hormones (Smith, 1930; Clermont and Morgentaler, 1955; Cler- mont and Harvey, 1965; Russell and Clermont, 1977). The hypophysectomy model is generally employed with nonseasonal breeders but has been rarely used in sea-

sonal breeders because the photoperiodic changes in seaonal breeders can be used to produce regression of the testis. There is virtually no information available to ascertain whether seasonal regression and hypophysectomy produce similar results. Both hypophysectomy and short-photoperiod exposure are known to reduce pituitary secretions, but it is not known if the manifestations of seasonal regression are the same as those seen after hypophysectomy. Moreover, it is not known if the effects of hypophysectomy in the seasonal breeder are equivalent to those in the nonseasonal breeder. These gaps in our knowledge form the objectives of the present study in which we target the Sertoli cell for investigation.

In addition to the aforestated objectives, it was deemed important to determine the changes manifest by the hamster Sertoli cell a t a very early time period after hypophysectomy when the functional deficits of the testis (germ cell degneration) were first observed. We wished to know if hypophysectomy-related changes are the same as those manifest during early seasonal regression and if these changes are correlated with specific endocrine parameters. Also in making these determinations, it was hoped that we will be able to determine structural parameters of the Sertoli cell that are most sensitive to the hormone deprivation produced by hypophysectomy.

MATERIALS AND METHODS Animals

Adult, male golden (Syrian) hamsters of the LAK: LVG (SYR) strain, 70-75 days of age, and weighing 100-110 g were obtained from Charles River Labora- tories, Inc. (Wilmington, MA). The animals were housed in the vivarium facility and exposed to a long photoperiod (16 hours of light, 8 hours of darkness). Animals were provided with food ad libitum, and a 5% sucrose solution was provided instead of water. The animals were divided into two groups with five animals in each group. All the animals were hypophysectomized by the supplier via the parapharyngeal route and the absence of the pituitary was verified at the time of sacrifice by examining the sella turcica. One group consisted of the short-term hypophysectomized hamsters (N = 5) that were sacrificed 6 days posthypophysectomy and the other group consisted of long- term hypophysectomized hamsters (N = 5) that were sacrificed 20 days posthypophysectomy. Data from the hypophysectomized animals were compared to data from the photoperiodic hamsters published in earlier

EFFECTS OF HYPOPHYSECTOMY ON HAMSTER SERTOLI CELLS 515

studies from this laboratory by Sinha Hikim et al. (198810,1989b). (Our rationale for using historical data for comparisons was that the time-consuming nature of such studies prohibited use of new control groups. The experimental groups that comprised five animals in each of two experimental groups alone took 2 years to analyze with the morphometric techniques chosen. Personnel changes did not allow the same individual to perform morphometric procedures on all experimental and control groups. It is fully realized that optimally, the control groups should have been performed at the same time as the present observations to reduce the possibilities of extraneous factors influencing the comparisons reported.) The data from hamsters hypophysectomized for the short term were compared to the intact long-day hamster data while the data from the long-term hypophysectomized animals were compared to the short-day hamster data.

Blood Collection and Specimen Preparation Fifteen minutes prior to sacrifice, the animals were

injected intraperitoneally (i.p.1 with heparin (130 IU/ 100 g body weight; Russell et al., 1990) following which the animals were anesthetized with Nembutalm. Under anesthesia, blood samples were withdrawn by cardiac puncture and collected in tubes containing ethyldiami- netetraacetic acid (EDTA). The plasma was separated by centrifugation at 4°C and frozen (at -2OOC). The right testis was removed and weighed (fresh testis weight) and the volume was determined by water displacement (Steer, 1981). The specific gravity was determined by dividing the testicular weight by the volume (Steer, 1981). The testis was then decapsulated and the tissue cut into three parts. Each portion was then weighed and placed in polypropylene tubes. The tissue was rapidly frozen in a mixture of dry-ice and acetone and stored frozen at -70 to -80°C until receptor and tissue testosterone assays were performed. A total of five fresh right testes were also obtained from intact hamsters kept in long photoperiod (14L: lOD) for FSH receptor assay to be run simultaneously with the tissue from hopophysectomized hamsters for the purpose of comparison.

The left testis was then perfused-fixed via the cardiac route with 5% glutaraldehyde in a 0.05 M cacodylate buffer (pH 7.4) preceded by a brief saline wash (Sprando, 1990). The paired seminal vesicles were removed and weighed to verify completeness of hypophysectomy. The fixed testis was cross-sectioned into -1 mm thick slabs. One slab each from proximal, distal, and equatorial regions were further diced into 1 mm cubes and immersed in the same fixative for an addi- tonal hour. The tissues were placed in cacodylate buffer overnight at 4°C and then postfixed in 1% osmium: 1.25% potassium ferrocyanide mixture (Russell and Burguet, 1977), dehydrated in a graded series of etha- nols, and embedded in Araldite (CY 212). At embedding, the blocks were oriented such that cross-sections of seminiferous tubules could be produced.

Shrinkage was determined by measuring the diameter of testicular cross-sectional slabs in resin taken from each animal and compared to the length of the same axis before secondary fixation, dehydration, and embedding according to Sinha Hikim et al. (1988b) and values were found to be comparable since the same technique was utilized for tissue preparation. Thus, to

correct for shrinkage due to fixation, dehydration, and embedding, a correction factor of 1.158, used by Sinha Hikim et al. (1988131, was adapted and used to correct all volumetric measurements. Since comparisons were made to hamster tissues from a different study, it was deemed important to correct for investigator differences in the performance of methodologies. The Sertoli cell nuclear volume was measured in this study in the same long-day hamsters utilized in the study by Sinha Hikim et al. (1988b) by nuclear reconstruction of five nuclei from each animal used by Sinha Hikim et al. (198813) and a mean nuclear volume was obtained for each animal. Twenty-five nuclei were measured. The mean volume thus obtained for the group of animals was compared to the published nuclear volume (Sinha Hikim et al., 198813) and found to be approximately 1.4 times larger. In order to compare results from the present study and that of Sinha Hikim et al. (1998b, 1989b), a factor of 0.705 was then applied to all data from the hamster to correct for differences in investigator measurement techniques.

The tissue embedded in blocks was sectioned using a Sorval MT-1 ultramicrotome at a microtome setting that has been previously measured to be 0.92 pm in thickness (Sinha Hikim et al., 198813) for serial section reconstruction and for morphometric analyses at the light microscope level. The sections were stained with 1% toluidine blue and mounted on glass slides and examined under a binocular Microphot-FX (Nikon) microscope equipped with a bright field condenser.

For electron microscopic studies, thin sections (sil- ver-gold) were obtained using the Reichert ultramicrotome (Ultracut-E, Reichert-Jung, Wien, Austria) and a diamond knife and the sections were mounted on Form- var-coated slot grids. These sections were poststained in uranyl acetate and lead citrate and examined under a Hitachi H500H transmission electron microscope (Hitachi, Tokyo, Japan). Staging criteria of Leblond and Clermont (1952) were used to identify stages of the hamster spermatogenic cycle.

Morphometric Procedures (Light Microscopy) Accepted morphometric procedures (Weibel and Bo-

lender, 1973; Elias and Hyde, 1980; Mori and Chris- tensen, 1980) were employed to obtain quantitative information on the testis specifically the Sertoli cell and its organelles in the two groups of hypophysectomized hamsters. Volume density and volume determinations

Point counting methods (Weibel, 1969; Weibel and Bolender, 1973; Mori and Christensen, 1980) were used to measure volume density of the testicular components such as seminiferous tubules, seminiferous epithelium, tubular lumen, interstitiurn, Leydig cells, blood vessels, and macrophages. Sections were collected at random, 10 sections per animal, and examined under the binocular Microphot-FX (Nikon) microscope equipped with a Hoffman bright field condenser, a x 40 objective, and a x 10 eyepiece. A square lattice containing 441 (21 x 21) intersections, was used as test points. The number of “hits” on the important structures was counted making sure that there was no overlap of the sections. Volume densities (V,) of testicular components were then determined by dividing the number of “hits” on each structure (Pi) by the total

516 S. GHOSH ET AL.

number of points over the tissue (PT). The volume percent of each component was calculated from the respective volume densities by multiplication by 100 (Chris- tensen and Peacock, 1980). The volumes (V) of each component were then determined by multiplying the volume densities (V,) by the whole testis (fresh) volume as represented by the formula

v = v, x v,. Seminiferous tubule length and diameter

Seminiferous tubules were selected at random to measure the diameter of the tubules across the minor axis of their profiles (Wing and Christensen, 1982). An ocular micrometer calibrated with a stage micrometer under a x 10 objective was used for this purpose. The total length (L) of the seminiferous tubule was then determined using the standard equation, L = Vvlm2 (Wing and Christensen, 1982), where r represents the average radius of the tubule, and V, the tubular volume per unit volume of the testis tissue as measured by point counting. The r and V, values were determined as mentioned above. The absolute length was then calculated by multiplying by the testicular volume (VT).

Sertoli cell and germ cell nuclear volumes and numerical densities and Serto1i:germ cell ratio

Five nuclei per animal per group were selected for serial reconstruction for measuring the volume of individual nuclei of Sertoli (stage VII) cells. Twenty serial sections were made for each animal. A single, middle section (- 10th) was then selected from the series of serial sections and the nuclei with the largest profile were selected at x 1,000. The rationale for selecting nuclei with the largest profile diameters was that these nuclei are sectioned through their real diameter allow- ing them to be traced readily from the middle section of the serially sectioned series. Five such selected nuclei were drawn using a camera lucida attachment. The same procedure was repeated for all the other sections in the series containing the selected nuclei. The actual magnification of the camera lucida drawings was then determined with a stage micrometer a t x 1,000. The individual volumes of nuclear profiles were calculated using the formula:

nuclear area (pm') x mean section thickness (pm) v,,(pm3) = (magnification')

The individual volumes of the nuclear profiles (V,J in serial sections were added to obtain the individual nuclear volume (V,). The area of the nucleus was determined with a Numonics (Model 1224) electronic digi- tizer, the accuracy of which was periodically checked using graphs of known areas. The principle for deter- mining the thickness for electron microscope sections (Russell et al., 1983) was applied to determine the mean section thickness for light microscope serial sections. This measurement was verified by earlier studies (Sinha Hikim et al., 1988b).

The numerical density (number of a particular cell type per unit volume of testis) of Sertoli and germ cells was measured by dividing the volume density of the nucleus by the individual volume of the nucleus (Kaler and Neaves, 1978; Budd, 1982). The numerical density of Sertoli cells (N,) was obtained using the formula:

N,S = V,S,NS, x 10''

where N,S is the numerical density of Sertoli cells, V,S, is the volume fraction of Sertoli cell nucleus in the testis, and VS, is the mean Sertoli cell nuclear volume (pm3). A value of lo1' was used to convert cm3 to pm3. The absolute number of Sertoli cells was determined by multiplying the number of cells per unit volume of the seminiferous tubule by the volume density of the seminiferous tubule. This gave the number of cells per unit volume of the testis [Russell et al., 19881. The absolute number of Sertoli cells was also determined by multiplying the value per unit volume by the testicular volume (fresh testis).

The numerical density of the germ cells was determined using the same principles of morphometry as described above. The volume density of the germ cell nuclei was measured by point counting using the same lattice. The number of germ cells (preleptotene and pachytene spermatocytes and round spermatids) per unit volume of seminiferous tubule was also recorded for testis of both groups. Since germ cells have a nearly spherical nucleus, the volume of an individual nucleus (V,) was calculated from the mean diameter (D) of the nucleus using the standard formula:

V, = 116 nD3 (Steer, 1981).

The largest diameter (D) was obtained from serial sections by direct measurement of its short axis at x 1,000 magnification using an ocular micrometer. Nuclei were measured in each section and the profile diameter that was maximum was used in the formula. The accuracy of this method has been verified by an earlier study in our laboratory (Sinha Hikim et al., 1988b). The number of germ cells per Sertoli cell (Serto1i:germ cell ratio) in the two groups was obtained by comparing average cell numbers (germ cells with Sertoli cells) in a unit volume of the seminiferous tubule (stage VII).

Morphornetric Procedures (Electron Microscope Studies)

Montage preparations To determine the fine changes in Sertoli cell mor-

phology, morphometric quantitation of the cell and its organelles was performed at the electron microscope level. Round tubules (at stage VII) were selected to examine Sertoli cells. The selection was random from each group of animals (n = 5). Sections were photo- graphed at x 4,800 or low magnification (negative magnification) and the negatives enlarged ( x 13,000) to construct a montage. The entire tubule was divided into portions of a clock as described by Sinha Hikim et al. (1989b) from which segments (wedge 118th of the entire tubule) were selected randomly. Photographs were taken starting from the base and then progress- ing toward the center of the imaginary clock. Lastly two lines were drawn on the composed montage from the center to the basal area of the tubule segment to demarcate a pie-shaped wedge to represent an even distribution of the tubule as a whole. For high magnification morphometry the above methods were employed to construct a portion (i.e., l i l6 th of the whole tubule) of the same wedge constructed at low power at a magnification of x 9,000 (negative magnification), and the negatives enlarged ( x 24,000) to construct a montage to quantify other intracellular structures re- solvable a t that magnification.


Volume measurements Montages constructed from the low magnification

micrographs in each group of animals were used to determine volume fractions of the Sertoli cell nucleus from which the cell volume was determined. Transpar- ent overlay with point grid system was used to measure volume densities as described above. The accuracy of this method using the montages was checked by comparing the volume density of the Sertoli cell nuclei within the wedge-shaped montage with the values obtained using a whole tubular profile a t the light microscope level as previously described by Sinha Hikim et al. (1989b). The individual Sertoli cell volume (Vsc) was calculated from the nuc1eus:cell ratio (PJP,) mul- tiplied by the volume of the nucleus Vsn) determined a t the light microscope level. The nuc1eus:cell ratio was obtained by point counting from the number of points falling on the nuclei (P,) and on whole cells (P,). The volume of the cytoplasm of the Sertoli cells (Vscy) was determined by subtracting the nuclear volume (Vsn) from the cell volume (Vsc).

Mitochondria, Golgi apparatus, rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), ectoplasmic specializations (ES), multivesicular bodies (MVB), lysosomes, and lipid droplets were Ser- toli cell organelles measured using the formula:

where Pi stands for the number of points on the organelle of interest and P,, stands for the total number of points counted over the Sertoli cell cytoplasm. The results were expressed as a percentage of the Sertoli cytoplasmic volume or V,% by multiplying the volume densities by 100. The absolute volume for each of the organelles was calculated by multiplying the volume densities (V,) by the Sertoli cytoplasmic volume (Vcy) for each individual animal in each group.

Surface densities and surface areas The low magnification montages of the stage VII tu-

bules were used to determine the surface density of the Sertoli cell plasma membrane. The Sertoli-Sertoli junc- tional area was used as a demarcation of the basal, and adluminal compartments of the cell and the surface densities of the plasma membrane in each of these areas was determined separately. The montages constructed from high magnification micrographs were used to measure the surface densities and surface areas of the Sertoli cell organelles. A multipurpose grid composed of straight lines (length of test line = 1.0 cm) was used over the tubular montages for this purpose. Sur- face densities (S,) of cell organelles such as mitochondrial inner and outer membranes, and Golgi apparatus were measured using the formula:

S, = 4IiPTZ (km2/p,m3)

where I is the number of intersections the test lines make with the surface of interest, PT is the number of “hits” over the cell cytoplasm, and Z is the average length of individual lines in terms of the scale of the micrograph (at the magnification being used for analysis). From this information, the surface area (S) was determined (km2) using a formula S = (S,) x VS,,, where S, is the surface density of the organelle and VS, represents the volume of the Sertoli cell cytoplasm.

Volume density (V,) = P,IP,,

The surface densities of structures that have an an- isotropic arrangement were measured using the Merz Curvilinear (length of test line = 2.0 cm) test systems (Weibel, 1979). Structures such as SER, and RER, and ES were measured using this system. The formulas for volume and for surface density described above were used. The surface areas were calculated from the surface densities. The surface area for each Sertoli cell compartment was determined by multiplying the respective surface densities by the Sertoli cell volume. The absolute surface area of each cell organelle measured was determined by multiplying the surface densities by the individual Sertoli cytoplasmic volume.

Endocrine Determinations Testosterone assay

The concentrations of testosterone in plasma as well as in testicular homogenates were measured by radio- immunoassay (RIA). Prior to tissue testosterone assay, testicular tissue was rapidly thawed and homogenized in 5 ml of cold distilled water. Aliquots of this homog- enate were then used for testosterone assay by RIA methods. The assay was performed as described by Chandrashekar and Bartke (1988). Testosterone was extracted with anhydrous ether (Aldrich chemical, Mil- waukee, MI). The antibody (GDN S250) used was de- veloped against testosterone and supplied by Dr. G.D. Niswender, Colorado State University. The cross reac- tivity of this antibody is as follows-26.0% with dihy- drotestosterone, 1.51% with androstenedione, and 0.14% with progesterone. The tracer used was 1,2,6,7,3H (TI (New England Nuclear Net 387). The supernatants containing the bound fraction were counted in a Packard-Tricarb Scintillation counter. The standard curve on a log vs. logit scale was linear between 5 and 500 pg/tube. Recovery of internal tacer was 88-92%. Sensitivity of this assay was 5 pgltube, and the intrassay and interassay coefficients of variability were (10% and <15%, respectively. FSH and PRL assays

Plasma levels of FSH and PRL were measured by RIA methods. Briefly, National Institute of Diabetes and Digestive and Kidney Diseases (N1DDK)-r-FSH- RP-2 and hamster prolactin (haPRL) were used as ref- erences for assaying FSH and PRL, respectively. The antisera used were NIDDK-anti-rFSH-S-11 and ly- ophilized rabbit anti-haPRL for FSH and PRL assays, respectively. The tracers used in this study were NIDDK-rFSH-1-7 for FSH and haPRL for PRL. The standard curve for FSH ranged from 0.0488-25 ng/ml and for PRL ranged from 0.0168-2 ng/100 ~ 1 . The sen- sitivities of the assay were FSH, 0.098 ng/ml; PRL, 0.039 ng/ml. PRL was measured in a separate assay. The intrassay coefficient of variation was 0.2% for FSH. The intrassay coefficient of variation for PRL was 3.9%.

Preparation of testis light membrane fraction Testicular tissue was thawed, homogenized in ice-

cold 10 mM Tris-HC1 buffer (pH 7.2) containing 1 mM MgC1, and 0.001% NaN,, and centrifuged for 15 minutes at 600g. The supernatant was collected and the pellet resuspended in Tris-HC1 buffer and centrifuged a second time for 15 minutes at 600g. The pooled supernatants were then centrifuged for 1 hour a t 40,OOOg.

518 S. GHOSH ET AL.

The supernatant was decanted and the pellet (containing the light membrane fraction) was resuspended in Tris-HC1 buffer. Protein concentration of the light membrane fraction was determined by Bradford's method (Bradford, 1976) using bovine serum albumin (BSA) as standard. The light membrane fraction was divided into 500 pl aliquots and stored a t -70°C until used in hormone binding assays.

FSH receptor binding assay FSH (LER-1781-2; 4,000 IU/mg) was radioiodinated

by the modified lactoperoxidase method (Fletcher et al., 1982). Radioiodinated hFSH was separated from free iodine by polyacrylamide gel electrophoresis as previously described (Schneyer et al., 1986). The specific activity of the iodinated hFSH, determined by self- displacement (Calvo et al., 1983), ranged between 25- 30 pCi/pg. Approximately 30% of total counts added to excess receptor was specifically bound.

Receptor binding assays were performed as described by Grasso and Reichert (1989). Briefly, 200 pl aliquots of the membrane fractions were incubated in polypropylene tubes (12 x 75 mm) for 22 hours a t 4°C in the presence of 2.5 ng 1251 [hFSH] and 4 pg ovine FSH (LER-1996-S,200 IUlmg). At the end of the incubation period, FSH receptor complexes were separated from unbound FSH by precipitation with 0.5 ml 0.2% (wtl vol) gamma-globulin and 0.9 ml 25% (wtlvol) polyeth- ylene glycol (PEG) in phosphate-buffered saline (PBS) (pH 7.0) a t 3,500g (Sorvall RC-3, HG-4L rotor) for 40 minutes. Supernatants were aspirated and radioactiv- ity in the pellets was counted in an LKB-Walac Clini- Gamma counter (LKB, Rockville, MD) with an effi- ciency of 85% for 1251.

Determination of FSH receptor number per cell The average number of receptors per Sertoli cell was

then derived by the method described by Sinha Hikim et al. (1989a) for LH receptors, using the following formula:

conc. (fmol/g testis) x 6.023 x 10' Sertoli cell number / g testis

Receptor no./cell =

where 6.023 x 10' is Avogadro's number which was used to obtain the total number of FSH receptor binding sites per gram of testis.

Sfafisticai Evaluation Since one of the objectives of this study was to com-

pare hypophysectomy with seasonal regression, a Stu- dent's t-test was used to compare the published long- day intact hamster data with data from the short-term hypophysectomized hamsters, and the short-day data with the long-term hypophysectomized hamster data obtained in this study. P < 0.05 was considered to indicate significant difference between groups. Correla- tion coefficients between the hormone levels and the morphometric parameters were determined using the StatView 512 + TM program for the Macintosh. For de- termining the correlation coefficients, the hypophysectomized groups were compared to the published data from our laboratory (Sinha Hikim et al., 1989b) for the long-day hamster group.

RESULTS

Perfusion fixation resulted in optimum preservation of the testis structure for morphometric studies (Figs. 1, 2). The basic data obtained from hamsters hypophysectomized for the short term (6 days) and long term (20 days) are shown in Table 1. In addition, all hypophysectomized data were compared statistically with the corresponding published data from long-day (control) and short-day (photoperiodic manipulations) animals as determined by Sinha Hikim et al. (198813, 1989131, data which are duplicated in this report.

Short-term hypophysectomy resulted in a significant decrease in testes weight as compared with that of the long-day animals (P < 0.001; 70%). (Percentage figures in parentheses represent decreases or increases mentioned in the accompanying statement.) There was a significant decrease in testes weight after long-term hypophysectomy that was significantly lower than the short-day animals (P < 0.001; a decrease of 53%) as well as the short-term hypophysectomized (P = 0.001; a decrease of 84%) animals. The mean weight of the paired seminal vesicles decreased significantly (P < 0.005; 75%) in long-term hypophysectomy as compared with short-term hypophysectomy.

Testicular Parameters The morphometric data pertaining to major testicu-

lar compartments in short- and long-term hypophysectomized hamsters are shown in Table 2. Compared to the reported values for intact, long-day hamsters, the tubule diameter (P < 0.001; 38%) as well as the length of the seminiferous tubule (P < 0.05,25%) was found to have decreased significantly after short-erm hypophysectomy. Long-term hypophysectomy produced a significantly smaller tubule diameter (P < 0.01; 21%) and resulted in a decrease in the length of the tubule (P < 0.05; 34%) compared with those measured in short-day animals. After long-term hypophysectomy, the tubule diameter (P < 0.001; 51%) and tubular length (P < 0.005; 44%) were significantly decreased as compared with animals hypophysectomized for a short term.

Table 2 shows the volumes of testicular compartments in the short-term and the long-term hypophysectomized hamsters. Short-term hypophysectomy resulted in a significant decrease in seminiferous tubule volume (P < 0.001; 70%) and in the volume of the tubular lumen (P < 0.001; 87%) in comparison with the respective volumes in the long-day hamster group. Long-term hypophysectomy resulted in a significant decrease in seminiferous tubule volume when compared with either short-day (P < 0.001; 58%) or short- term hypophysectomized animals (P = 0.001; 87%). The tubular lumen volume decreased significantly after long-term hypophysectomy as compared with short- term hypophysectomized animals (P < 0.005). There was no significant difference in tubular lumen volume in short-day animals as compared with long-term hypophysectomized animals (P > 0.05), although the lumen was not present in any long-term hypophysectomized animals; whereas, some (two out of five) short- day testes showed tubular lumena. Long-term hypophysectomy resulted in a significant decrease in epithelial volume as compared with short-term hypophysectomized animals (P = 0.001; 86%).

Fig. 1. A Seminiferous tubules at mid-cycle (stage VII) in a short- term hypophysectomized hamster. The tubules show mostly normal appearing viable cells. Among viable cells, the epithelium also shows various germ cells in the process of degeneration. Among them are pachytene spermatocytes, step 7 spermatids, and elongated sperma-

tids (step 16). Vacuoles are seen near the basal region of the tubules. x 660. B: Seminiferous tubules in hamsters hypophysectomized for 20 days. No tubular lumen is seen. The tubules shown are small and mainly contain Sertoli cells and basal compartment cells. x 660.

After short-term hypophysectomy there was a significant decrease in the volume of the interstitium (P < 0.001; 69%), including a decrease in volume of the Ley-

dig cells (P < 0.001; 81%), the volume of blood vessels (P < 0.001; 75%) and macrophages (P < 0.05; 42%) as compared with the intact long-day group. Long-term

Fig. 2. A Electron micrograph of Sertoli cells in a short-term hypophysectomized hamster. The nucleoli appear small. The cytoplasm shows the products of digestion of germinal cells (arrows). Numerous small lipid droplets (L) are also seen. Other structures indicated are

Golgi complex (GI, and ectoplasmic specialization (ES). x 9,600. B: Electron micrograph showing several Sertoli cells from a 20 day hypophysectomized hamster. The apical cytoplasm is crowded with lipid inclusions. Ectoplasmic specialization is sparse. x 9,600.

hypophysectomy, as compared to gonadally-regressed short-day animals, resulted in a significant decrease in interstitial volume (P < 0.001; 36%), total Leydig cell

volume (P < 0.01; 35%), total blood vessels volume (P < 0.001; 62%), and total macrophage volume (P < 0.05; 33%). There was also a significant decrease in the total

EFFECTS OF HYPOPHYSECTOMY ON HAMSTER SERTOLI CELLS

TABLE 1. Basic data on hamsters hypophysectomized for the short term (6 days) and long term (20 days)

52 1

Parameters'

Long-day2 Short-term intact hypox

(n = 5) (n = 5)

Long-term hypox

(n = 5)

Short-day2 intact

(n = 5) Body weight (g) 160.40 t 7.50 96.96 * 1.69* 107.94 t 3.72*.** 159.30 2 9.70 Fresh (right) testis weight (g) 1.74 t 0.10 0.51 2 0.09* 0.08 t 0.004*,*** 0.18 2 0.02 Fresh (right) testis volume (cm3) 1.66 t_ 0.09 0.49 +- 0.09* 0.08 * 0.003*,*** 0.17 * 0.02 Specific gravity of fresh testis 1.04 t 0.003 1.04 * 0.004 1.09 2 0.03**** 1.03 2 0.08

'Mean -t SE. 'Data from Sinha Hikim et al. (1988). "Means differ significantly from the photoperiodically manipulated animals (short-term vs. long-day; long-term vs. short-day) with P < 0.01. +*P < 0.01; ***P = 0,001; ****P < 0.05 (these means differ significantly from the short-term hypophysectiomized animals).

Short-term Hypophysectomized hamsters 3 A

H Mitochondria

Golgi complex

SER

Ed- 0 Ectoplasmic specialization

Lipid

El Lysosomes

Multivesicular bodies

Cytoplasmic matrix

Long-term Hypophysectomized hamsters B

Mitochondria

Golgi complex

SER

Elm Ectoplasmic specialization

Lipid

Lysosomes

H Multivesicular bodies

Cytoplasmic matrix

Fig. 3. Volumetric composition of the Sertoli cell cytoplasm in short-term (A1 and long-term (B) hypophysectomized hamsters. The pie graphs are proportional to the diameter of the cytoplasm of an artificial rounded cell.

volume of the interstitium (P = 0.001; 44%), Leydig cells (P < 0.05; 30%), blood vessels (P < 0,005; 78%), and macrophages (P < 0.001; 64%) in long-term hypophysectomized animals as compared to those hypophysectomized for a short-term duration.

Short-term hypophysectomy significantly (P < 0.05) lowered (31%) the mean number of Sertoli cells from that reported for the long-day hamster (73.88 x lo6). The mean number of Sertoli cells was not significantly different in the long-term hypophysectomized animals

522 S. GHOSH ET AL.

TABLE 2. Morphometric data' on various testicular parameters in hamsters hypophysectomized for the short term and long term

Parameter Tubule diameter (ym) Length (meterdhestis Percent volume of testicular components

Seminiferous tubule Seminiferous epithelium Tubular lumen Interstitium Leydig cell Blood vessels

Volumehestis Seminiferous tubule (ml) Seminiferous epithelium (ml) Tubular lumen (p1) Interstitium (p1) Leydig cell (pl) Blood vessels (y1) Macrophages (pl)

Sertoli cell number ( x 106)/g of testis Sertoli cell number ( x 106)/testis

Long-day (intact)' (n = 5 )

275.36 t 5.29 25.92 t 0.63

92.55 f 0.84 NA3

14.37 t 0.37 6.22 t 0.27 1.42 2 0.03 1.49 f 0.14

1.52 2 0.01 NA

238.54 * 6.14 103.25 t 4.48 23.57 t 0.49 24.73 t 2.32 4.15 t 0.49

44.51 t 3.22 73.88 * 5.34

Hypox-6 days (n = 5 )

170.32 t 14.75* 19.29 t 2.18*

92.49 * 1.41 86.06 f 1.82 6.43 * 1.14 7.52 t 1.41 1.15 t 0.03 1.31 t 0.14

0.46 t 0.09* 0.43 t 0.08

31.95 t 7.20* 32.18 k 2.85* 4.56 t 0.63* 6.16 t 1.06* 2.40 i 0.25*

137.71 t 52.76 50.89 t 7.98*

Hypox-20 days (n = 5)

Short-day (intact)' (n = 5 )

84.08 f 1.15l,** 10.70 t 0.72*****

76.46 * 0.64 76.46 t 0.64

0 2 0 23.54 f 0.64 4.12 t 0.36 1.76 t 0.16

0.06 t 0.02*.**** 0.06 f 0.002****

0 t o*** 18.15 t 0.66*~**** 3.18 t 0.31******* 1.35 t 0.12*9*** 0.86 * 0.06****

803.05 t 47.29 62.00 t 4.25

107.23 t 6.63 16.36 2 1.87

83.33 t 1.46 NA

1.66 t 0.81 16.59 t 1.39 2.88 t 0.21 2.11 t 0.30

0.14 2 0.002 NA

2.82 2 1.37 28.20 t 1.39 4.89 t 0.35 3.58 t 0.50 1.29 2 0.13

469.98 t 80.51 79.89 * 13.68

'Means ? SE. 'Data from Sinha Hikim et al. (1988b). 3NA, data not available. *Means differ significantly from the photoperiodic animals (short-term vs. long-day; long-term vs. short-day) with P < 0.05. **P < 0,001; ***P < 0.005; ****P = 0.001; *****I' < 0.05 (these means differ significantly from short-term hypophysectomized animals).

TABLE 3. Germ cell: Sertoli cell ratios in stage VII tubules in hvDoDhvsectomized hamsters'

Long-day Short-term Long-term Short-day' intact hypox hypox intact

Cell types (n = 5 ) (n = 5 ) (n = 5 ) (n = 5 ) PreleDtotene sDermatocvtes 2.27 t 0.12 2.85 f 0.43* 0.82 t 0.09*,** 0.96 t 0.07 Pachytene speimatocytes 2.46 t 0.09 1.77 f 0.62 0 t o*.*** 0.20 t 0.07 Round spermatids 8.17 t 0.56 3.89 t 1.12* 0 t o**** 0.04 t 0.02

'Ratios derived from the average cell number per unit volume (cm3) of the seminiferous tubule (stage VII). Mean t SE. 'Data from Sinha Hikim et al. (198813). *Means differ significantly from the photoperiodic animals (short-term vs. long-day; long-term vs. short-day) with P < 0.05 **P < 0,001; ***P = 0.01; ****P < 0.005 (these means differ significantly from short-term animals).

as compared to short-day (P < 0.3) and short-term animals (P = 0.25).

Serto1i:Germ Cell Ratio The germ cel1:Sertoli cell ratios in stage VII of the

spermatogenic cycle are shown in Table 3. The ratio of preleptotene spermatocytes to Sertoli cells was not significantly different after short-term hypophysectomy as compared with long-day animals (P < 0.5). After long-term hypophysectomy, the ratio of preleptotene spermatocytes to Sertoli cells was not significantly different from short-day hamsters (P > 0.5), but significantly lowered as compared with animals hypophysectomized for a short-term (P = 0.001; 71%). The ratio of pachytene spermatocytes to Sertoli cells was not decreased significantly (P > 0.3; 28%) after short-term hypophysectomy as compared with long-day animals. In long-term hypophysectomy, the ratio was significantly lowered compared to short-day animals (P < 0.05) and animals hypophysectomized for a short term (P = 0.01). Pachytene spermatocytes were no longer present after long-term hypophysectomy. The ratio of step 7 spermatids to Sertoli cells was decreased significantly after short-term hypophysectomy (P < 0.01; 52%) as compared to the reported data in long-day

hamsters. After long-term hypophysectomy, the number was significantly lower than that seen after the animals have been hypophysectomized for a short term (P < 0.005) but was not significantly different as compared with short-day animals (P > 0.05). There were no step 7 spermatids after long-term hypophysectomy.

Sertoli Cell Volume and Surface Area Parameters (Tables 4 and 5)

There was no significant change in the nuclear volume, the cell volume, or cytoplasmic volume after short-term hypophysectomy as compared to intact hamsters maintained in a long-day environment (Sinha Hikim et al., 1988b, 198913). Compared to the 6-day hypophysectomized animals, the 20-day hypophysectomized animals showed a significant decrease in nuclear volume (P < 0.001; 58%), cell volume (P < 0.005; 82%), and cytoplasmic volume (P < 0.005; 85%). As compared with short-day hamsters, long-term hypophysectomy resulted in a significant (P < 0.005; 32%) decrease in nuclear volume but not in cell volume (P = 0.06) or cytoplasmic volume (P = 0.1).

Short-term hypophysectomy resulted in a significant (P < 0.05; 40%) decrease in total Sertoli cell surface


TABLE 4. Morphometric data on volumetric composition of hypophysectomized hamster Sertoli cells

Long-day Short-term Long-term Short-day intact hypox hypox intact

Component Parameter' (n = 5) (n = 5) (n = 5) (n = 5) Mitochondria V,% 5.48 t 0.33 5.05 f 0.232 4.97 t 0.03' 6.66 f 0.70

V 203.42 t 17.93 185.73 f 39.52*** 26.07 2 2.41** 54.22 f 10.37 Lipid V,% 0.72 f 0.11 1.41 t 0.78 18.00 f 2.11 4.10 2 1.96

V 27.11 f 4.44 35.62 f 12.75 90.81 f 5.61** 32.79 t 13.42 Lysosomes V,% 0.60 f 0.08 3.39 f 1.21 6.24 f 1.06 3.78 t 0.90

V 22.18 * 2.92 105.90 f 27.42 33.83 ? 7.57*** 34.49 + 12.07 Golgi complex V,% 1.27 f 0.32 0.89 2 0.41 0.85 t 0.33 1.42 f 0.29

V 45.45 2 9.63 29.20 t 13.55 4.10 f 1.25 11.69 f 2.63 SER V,% 7.48 * 0.85 10.58 f 0.84 4.95 f 0.26 7.61 2 0.51

V 286.87 f 53.16 393.41 2 86.40 26.27 f 2.97** 64.40 f 14.95 RER V,% 1.28 t 0.23 1.26 f 0.40 1.44 t 0.28 0.78 * 0.13

V 45.96 f 7.76 37.50 t 5.90 7.68 + 1.74**** 7.23 f 2.62 ES V,% 1.94 * 0.20 0.90 t 0.27 0.59 f 0.13 1.04 f 0.29

V 73.38 t 11.84 27.18 2 3.93 3.31 f 0.86**** 10.35 f 5.16 MVB V,% 0.48 f 0.09 0.10 t 0.04 0.17 t 0.03 0.19 f 0.03

V 17.15 2 2.89 2.73 f 1.15 0.76 f 0.11 1.54 + 0.28 Nucleus V,% 11.87 f 1.23 14.13 f 1.96 30.11 f 1.57 28.32 f 3.04

V 502.75 f 26.77 529.76 ? 42.58 233.76 f 16.19*,**** 331.09 f 19.18 Cell V 4,233.51 f 330.42 4,160.49 t 790.85 748.56 t 56.93** 1,168.76 f 186.57 Cytoplasm V 3,730.76 t 330.30 3,630.73 f 756.73 524.80 f 45.83** 837.67 f 168.89 'V,%, volume density expressed as a percentage of Sertoli cell cytoplasmic volume; V, absolute volume (pm3); SER, smooth endoplasmic reticulum; RER, rough endoplasmic reticulum; ES, ectoplasmic specializations; MVB, mutlivesicular bodies. 'Mean ? SEM. *Means differ significantly from the photoperiodic animals (short-term vs. long-day; long-term vs. short-day) with P < 0.05. **P < 0.005; ***P < 0.05; ****P < 0.001 (these means differ significantly from short-term animals).

523

area as compared to the intact long-day animals, re- sulting mainly from the decrease in the adluminal compartment surface area (P < 0.05; 47%), although the Sertoli cell basal compartment surface area was significantly increased (P < 0.005, 49%) from the values measured in long-day hamsters. Compared to the short-term hypophysectomized hamsters, the long- term hypophysectomized hamsters showed a significant decrease in the total Sertoli cell surface area (P = 0.005; 81%) including the basal compartment (P = 0.001; 60%) and the adluminal compartment surface area (P < 0.01; 85%). When compared to the short-day data, the cell surface area of the cell in the long-term hypophysectomized hamsters was significantly less (P < 0.05; 31%) as a result of the significant decrease in the surface area of the adluminal compartment (P < 0.01; 45%).

Sertoli Cell Organelle Volume Parameters (Figure 3 and Table 4)

In a comparison between 6 and 20 days posthypophysectomy groups, almost all organelle volumes decreased significantly (RER, P < 0.01; 79%; SER, P < 0.005; 93%; ES, P < 0.001; 88%; mitochondria, P < 0.005; 86%) except lipid which increased significantly (P < 0.005; 61%). The total volume of lysosomes decreased significantly (P < 0.05; 68%) which was due to a significant decrease in the volume of the secondary lysosomes (P < 0.01; 93%), and not primary lysosomes. The significantly increased volume of cytoplasmic vacuoles seen after short-term hypophysectomy (Ghosh et al., 1991) is significantly decreased after long-term hypophysectomy (P < 0.05; 98%).

A comparison of Sertoli cell organelle volumes in

short-term hypophysectomized hamsters to the intact long-day hamsters showed that there was no significant change in the volumes of the RER, SER, Golgi complex, lipid and mitochondria 6 days after hypophysectomy; however, there was a significant increase in the volume of the total lysosomes (P < 0.05; 79%). ES (P < 0.01; 63%) and MVB (P < 0.001; 82%) decreased significantly.

A comparison of long-term hypophysectomy with short-day exposed intact animals demonstrated no significant difference in the volumes of RER, total lysosomes, MVB, and ES although there was a significant decrease in the long-term hypophysectomized animals in the volume of SER (P < 0.05; 59%), motochondria (P < 0.05; 52%), and Golgi complex (P < 0.05; 65%). There was however, a significant increase in long-term hypophysectomized animals in the volume of lipids (P < 0.005; 64%) as compared with short-day animals.

Sertoli Cell Organelle Surface Areas (Table 5) In a comparison of short- and long-term hypophysec-

tomized hamsters, the surface areas for each of the Ser- toli cell organelles determined were significantly reduced in the latter (mitochondrial inner membrane: P < 0.005, 85%; mitochondrial outer membrane: P < 0.005, 86%; RER: P < 0.01, 72%; SER: P < 0.05, 90%; ES: P < 0.005,83%) except for the surface area of Golgi complex which was not reduced.

Following short-term hypophysectomy there was no sigificant change from the long-day results in the surface areas of any of the membraneous organelles except ES (P < 0.05; 55%) which decreased significantly. Re- sults of long-term hypophysectomy were not signifi-

524 S. GHOSH ET AL.

TABLE 5. Surface densities and surface areas of Sertoli cell organelles in hamsters hypophysectomized for 6 days (short-term) and 20 days (long-term)'

~

Long-da Short-term Long-term Short-day2 (intact) B

Component Parameter (n = 5) (n = 5) (n = 5) (n = 5) Mitochondrial S V 1.1 t 0.03 0.76 t 0.073 0.70 t 0.053

outer membrane S,/cell 4,180.20 t 451.40 2,714.86 ? 561.72 376.81 t 55.38** S,/a testis 1,860.60 ? 200.90 2,772.85 t 394.65 2.977.29 t 413.63

Mitochondrial inner membrane

Golgi complex

RER

SER

ES

Cell membrane

Basal compartment

Adluminal

Si/testis

S V S,/cell SA/g testis SA/testis

S V SA/cell SA/g testis S,/testis

S V S,/cell S,/g testis S,testis

S" S,/cell S,/g testis SA/testis

S V S,/cell S,/g testis S A 1 t e s t i s SA S,/g testis S,/testis

S A S,/g testis S,/testis S A

3,088.50 t 333.50

6,156.90 ? 725.80 2,740.40 ? 323.10 4,549.10 t 536.20

0.49 t 0.12 1,851.40 ? 565.70

824.10 t 251.80 1,368.00 t 417.90

1.67 t 0.39 5,926.70 ? 1,214.20 2,637.90 t 540.40 4,379.00 t 897.10

5.58 t 0.32 21,131.60 -C 2,806.30

9,405.70 +- 1,249.10 15,613.40 t 2,073.30

1.52 -C 0.16 5,746.10 t 1,027.00 2,557.60 t 457.10 4,245.60 t 758.70

13,273.70 t 1,320.50 5,908.10 t 587.70 9,807.40 t 975.60

598.30 t 41.20 266.30 t 18.30 442.00 2 30.30

12,675.40 ? 1,313.40

1.63 t 0.05 1i366.13 t 196.81

4,349.26 t 860.65 4,567.07 t 770.35 2,256.13 ? 380.55

1,540.43 t 531.39 1,944.00 t 634.47

960.34 t 313.43 1.75 t 0.70

5,006.67 t 992.86 7,766.22 c 4,120.35 3,836.51 t 2,035.45

8.42 t 0.80 32.416.07 -C 9,321.58 32.953.85 t 7,701.09 16,279.20 t 3,804.34

0.75 t 0.09 2,570.95 t 501.80* 2,975.65 ? 788.02 1,469.97 ? 389.28 7,904.51 t 1,679.87* 8,482.41 t 1,713.64 4,190.31 ? 846.54 1,169.93 t 125.64 1,614.28 ? 694.11

797.46 ? 342.89 6,734.47 ? 1,605.20*

1.22 t 0.08

0.44 ? 0.12

~~ ~.

229.25 t 31.85 1.19 c 0.10

636.12 t 197.14** 5,015.33 2 720.26

386.18 t 55.46 0.62 t 0.25

300.75 ? 99.16 2,457.52 ? 904.99

189.23 t 69.68 2.58 t 0.50

1,406.19 t 331.06*** 11,243.09 t 2,669.67

865.72 t 205.56 5.83 ? 0.69

3,148.04 ? 542.79**** 24,722.47 t 4,156.60

1,903.63 ? 320.06 0.80 t 0.16

445.62 t 118.53***** 3,513.85 ? 903.62

270.57 t 69.58 1,488.36 ? 129.96*2**

11,728.54 t 537.65 903.10 t 41.40 465.52 t 36.67*,******

3,755.33 t 427.46 289.16 ? 32.91

1,022.84 ? 151.60*,*** compartment S,/g testis 5,641.80 t 585.00 6,867.79 t 1,039.14 7,973.21 t 914.99

S,/testis 9,365.40 t 971.20 3,392.69 t 513.33 613.94 t 70.45 1,493.20 t 153.70

0.78 t 0.08 657.80 ? 152.10

3,091.70 t 713.50 525.50 5 120.20

1.23 t 0.13 1,063.50 t 270.90 4,998.30 c 1,270.80

849.70 t 214.10 0.76 t 0.03

636.20 t 128.20 2,989.90 t 601.20

508.30 t 101.30 0.78 t 0.05

691.30 2 185.40 3,249.20 t 869.60

552.30 t 146.40 7.41 t 0.29

6,339.90 t 1,458.20 29,796.60 t 6,838.90 5,065.40 t 1,151.90

1.58 t 0.30 1,498.10 t 563.40 7,040.90 ? 2,642.50 1,196.90 t 445.10 2,152.60 t 202.90

10,116.80 t 953.60 1,719.80 5 162.10

283.60 t 20.80 1,333.10 t 98.10

226.60 t 16.60 1,868.90 t 192.40 8.783.70 t 904.60

'S,, surface density (square microns per pm3 Sertoli cell cytoplasm); S, surface area (square microns per cell, square centimeter per gram testis, and square centimeter per testis, respectively); RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; ES, ectoplasmic specialization. 'Data from Sinha Hikim et al. (1989b). 3Mean ? SEM. *Means differ significantly from the photoperiodic animals (short-term vs. long-day; long-term vs. short-day) with P < 0.05. **P = 0.005; ***P < 0.01; ****P < 0.05; *****P < O.O05;******P < 0.001, (these means differ significantly from short-term animals).

cantly different from the short-day data in the membrane surface areas quantified.

Endocrine Findings (Table 6) The mean values of the plasma FSH and plasma tes-

tosterone in the 6-day hypophysectomized animals as compared with the intact long-day animals (Sinha Hikim et al., 1989b) declined significantly to 2.67 ng/ ml (65%, P < 0.005) and 0.44 ng/ml (84%, P < 0.001), respectively, from 7.7 ng/ml and 2.72 ng/ml reported (Sinha Hikim et al., 1989b). At 20 days posthypophysectomy, the levels of these hormones were not significantly different from the levels measured after 6 days. The average values of the plasma FSH and testosterone in these long-term hypophysectomized animals were also not significantly different from the short-day hamster data. Testicular testosterone showed a significant (P < 0.001) reduction (80.7%) in short-term hypophysectomized animals as compared with intact long-day animals.

The concentration of FSH receptors in the short-term hypophysectomized hamsters was 3.2 fmol/mg of protein which was not significantly different from the concentration of receptors in the long-day animals. The content of FSH receptors was significantly (P < 0.005) lowered after short-term hypophysectomy to 1,934 fmol per testis from 4,933 fmol per testis in the corresponding long-day group. The number of FSH receptors in the short-term hypophysectomized hamsters was 27.72 x lo3 per Sertoli cell corresponding to 23 receptors per each square micrometer of the basal compartment Ser- toli plasma membrane.

Correlation of Structural and Endocrine Parameters The intact long-day, short-term and long-term hy-

pophysectomized groups were used to correlate structural parameters with endocrine determinations. The volumes (Table 7) of some organelles exhibited a positive and significant correlation with plasma FSH (RER. r = 0.64, P < 0.02; ES: r = 0.85, P < 0.01; MVB:


TABLE 6. Endocrine parameters in hypophysectomized (6 days, short-term; 20 days, long-term) hamsters'

525

Long-day Short-term Long-term Parameter (n = 5) (n = 5) (n = 5) Plasma hormones2

T (ng/ml) FSH (ng/ml) PRL (ng/ml) LH (ng/ml)

Testicular steroid' T (ng/g testis)

FSH Receptors3 Concentration (fmolimg protein) Content (fmol/testis) Number ( x 103)/Sertoli cell Number/pm2 Sertoli cell

Plasma membrane Number/pm2Sertoli cell

Basal compartment membrane

2.72 ? 0.32 7.69 ? 1.29

2.53 ? 0.25

73.18 ? 3.79

2.99 t 0.26 4,932.75 f 301.00

NA

NA

NA

0.44 t 0.16* 2.67 t 0.62** 5.09 ? 1.39 0.78 t 0.34**

14.09 ? 4.68*

3.20 t 0.66 1,933.70 f 500.53**

27.72 t 11.5

2.78 t 0.44

23.18 * 5.09

0.36 ? 0.10 2.24 ? 0.17 6.49 ? 0.23 0.43 ? 0.24

NA4

NA NA NA

NA

NA 'Means ? SE. T, testosterone; FSH, follicle-stimulating hormone; PRL, prolactin; LH, luteinizing hormone. 'Long-day data from Sinha Hikim et al. (198913). 3Data represent the means of three of five animals. 4Data not available. *P = 0.0001; **P < 0.005 (these means differ significantly from long-day animals).

TABLE 7. Correlation coefficients and levels of significance for hormonal levels and Sertoli cell component volumes and surface areas in

hamsters* (n = 15; five long-day hamsters, five short-term hypophysectomized, and

five long-term hspophssectomized) ~

Correlation coefficients (r) Plasma FSH Plasma T

(nglml) Volume' (um3)/cell

Cell Nucleus Mitochondria Lipid Lysosomes SER RER ES Golgi Complex MVB

Cell Adluminal compartment Basal compartment 0 Mit. M. I. Mit. M.

Surface area (pm2)/ceH

SER RER ES

0.529 (<0.05)* 0.359 (NS)

-0.373 (NS) -0.30 (NS) 0.342 (NS)

0.64 (<0.02) 0.853 (<0.01) 0.48 (NS)

0.745 (<0.01)

0.622 (<0.02)

0.665 (<0.01) 0.685 (<0.01)

0.713 (<0.01) 0.701 (<0.01) 0.277 (NS) 0.505 (NS) 0.759 (<0.01)

-0.141 (NS)

0.328 (NS) 0.332 (NS) 0.399 (NS)

-0.487 (NS) -0.48 (NS) 0.112 (NS) 0.523 (<0.05) 0.802 (CO.01)

0.863 (<0.01) 0.42 (NS)

0.628 (<0.02) 0.654 (<0.01)

0.563 (<0.05) 0.52 (<0.05)

0.001 (NS) 0.456 (NS) 0.579 (<0.05)

-0.244 (NS)

Golgi complex 0.37 (NS) 0.163 (NS) 'Data from Sinha Hikim et al. (1989b). 'MVB, multivesicular bodies; 0. Mit. M., outer mitochondrial membrane; I. Mit. M., inner mitochondrial membrane; SER, smooth endoplasmic reticulum; RER, rough endoplasmic reticulum; ES, ectoplasmic specializations. *P values are in parentheses.

r = 0.75, P < 0.01) and also testosterone levels (RER: r = 0.52, P < 0.05; ES: r = 0.8, P < 0.01; MVB: r = 0.86, P < 0.01) while the cell volume (r = 0.53; P < 0.05) and mitochondria (r = 0.62; P < 0.02) showed a positive and significant correlation only with plasma

FSH level. Lipid and lysosome volumes were not significantly correlated with both plasma FSH and plasma testosterone levels. Other organelles including nuclear volume and volumes of SER and Golgi complex showed no correlation with either of the hormones measured.

The surface area (Table 7) of the cell and the adluminal compartment surface area showed a positive and significant correlation with FSH (r = 0.67; r = 0.69, respectively) and plasma testosterone (r = 0.63; r = 0.65, respectively). ES membrane surface area showed a positive and significant correlation with plasma FSH (r = 0.76; P < 0.01) and plasma testosterone levels (r = 0.58; P < 0.05). The mitochondrial outer membrane (r = 0.71) and the inner membrane (r = 0.7) showed a positive and significant (P < 0.01) correlation with the plasma FSH levels. The outer mitochondrial membrane (r = 0.56) and the inner membrane (r = 0.52) also exhibited positive and significant (P < 0.05) correlations with plasma testosterone levels.

The correlations between tissue testosterone levels and the volumes and surface areas of the Sertoli cell and its organelles were determined utilizing long-day and short-term hypophysectomized animals. Only the volumes of MVB (r = 0.87) and ES (r = 0.8) showed a significant (P < 0.01) and positive correlation with the tissue testosterone levels while the volume of the lysosomes showed a significant negative (r = -0.83; P < 0.01) correlation. The Sertoli cell volume, the nuclear and cytoplasmic volumes, and organelle volumes were not significantly correlated to the tissue testosterone levels. There was a significant negative correlation between the surface area of the Sertoli cell's basal compartment (r = -0.78; P < 0.01) and testicular testosterone levels and a significant positive correlation (r = 0.65; P < 0.05) between tissue testosterone and the membrane surface area of the adluminal compartment. None of the organelle surface areas showed a significant correlation with testicular testosterone levels although the surface areas of SER and Golgi membrane were negatively, but not significantly correlated.

526 S. GHOSH ET AL.

A correlation between Sertoli cell size and the organelle volumes and surface areas was performed for the intact long-day, short-term, and the long-term hypophysectomized hamsters. There was a positive and significant (P < 0.01) correlation between the average Sertoli cell volume and the volume of its organelles, including RER (r = 0.72), SER (r = 0.951, ES (r = 0.69), mitochondria (r = 0.98), and Golgi complex (r = 0.57; P < 0.05). A negative, but significant, correlation was seen with the volume of lipids (r = -0.72; P < 0.01). Similarly the surface areas of the mitochondrial inner membrane (r = 0.94) and the mitochondrial outer membrane (r = 0.91), the Golgi complex (r = 0.70), RER (r = 0.62; P < 0.02), SER (r = 0.92), and ES (r = 0.76) showed a positive and significant (P < 0.01) correlation with the average Sertoli cell volume.

DISCUSSION A major objective of the present study was to deter-

mine the quantitative structural changes in the Sertoli cell after hypophysectomy, especially those seen when a functional deficit in spermatogenesis was first evident. The results showed that while a t 6 days posthypophysectomy there was a marked decrease in testicular parameters in the tubular and intertubular compartments, there were virtually no changes in the Sertoli cell parameters. The Sertoli cell, which contains FSH and androgen receptors and is thought to be the mediator of FSH (Steinberger et al., 1974, Orth and Christensen, 1977) and testosterone (Sanborn et al., 1975, Grootegoed et al., 1977) action, showed few changes in short-term hypophysectomized hamsters at a time when the levels of these hormones were already maximally decreased. The Sertoli cell volume and nuclear volume did not change after short-term hypophysectomy, although the cell surface area decreased significantly at 6 days. It is not known if the reduced surface area is a primary effect of hormonal deprivation or is secondary to loss of germinal cell contact subsequent to germ cell degeneration. Since FSH is known to influence Sertoli cell shape in culture to increase its surface area (Sanborn et al., 1987; Tung et al., 1975), the lack of FSH may be responsible for the decrease in surface area noted.

At the ultrastructural level, short-term hypophysectomy resulted in no change in the volume of most Ser- toli cell organelles. The only organelle to increase in volume in short-term hypophysectomized animals was total lysosomes which increased 79% from the value in intact long-day animals. This can be attributed to the expected increase in germ cell degeneration and the appearance of secondary lysosomes, not primary lysosomes. The surface areas of virtually all subcellular components and organelles did not change significantly after short-term hypophysectomy (except ES which again may be secondary to loss in germ cells).

We are suspicious about one particular finding in the present study, namely that the number of Sertoli cells in hamsters hypophysectomized for 6 days decreased significantly from their number in the long-day animals. It has been established that Sertoli cells do not divide in the adult or hypophysectomized animals (Steinberger and Steinberger, 1977). They have been shown to remain unchanged in the hamsters exposed to short photoperiod (Sinha Hikim et al., 1988b) although

Johnson and Thompson (1983) have suggested seasonal variation in their numbers in horses. The unexpected decrease in the present study was not reflected in long- term hypophysectomized hamsters, so we suggested that the decrease reported in Sertoli cell numbers in short-term hypophysectomized hamsters is a false positive result associated with the manner in which Ser- toli cell numbers are obtained as the product of several factors. Numbers of Sertoli cells in studies of normal animals of the same species are extremely variable (De Jong and Sharpe, 1977; Hochereau-de-Reviers and Courot, 1978; Wing and Christensen, 1982; Orth et al., 1988; Kerr, 1988; Russell et al., 1988, 1990) also pos- sibly reflecting methodological peculiarities.

A second objective of this study was to determine if the Sertoli cell in an animal undergoing changes that mimic seasonal regression is like or unlike an animal that has been hypophysectomized. In the comparison of long-term hypophysectomized hamsters to short-day hamsters, our strategy was to compare groups that showed features indicating maximal regression had taken place, this in spite of variability in the time elapsed since the onset of the “condition.” In short-day photoperiodically regressed hamsters, the level of the hormones attained after full regression was statistically similar to that of hypophysectomized hamsters.

Changes in the testis and those specifically involving the Sertoli cell during photoperiod-induced testicular regression were found to be qualitatively similar to the changes that are seen in the Sertoli cell after hypophysectomy. However, hypophysectomy appeared to produce a slightly more severe regressive effect than short photoperiod. This was evident from examination of the testis microscopically and from examination of basic testicular parameters and germ cell numbers. For ex- ample, the tubular lumen, which is an indicator of fluid production by the Sertoli cell, is seen in some tubules from short-photoperiodic hamsters (Sinha Hikim et al., 1988b); whereas, after long-term hypophysectomy the lumen is completely absent. Com- parison of most parameters in long-term hypophysectomy and short-day animals showed no major differences. There was, however, a trend for a greater decrease in most parameters after long-term hypophysectomy. Lipid, which is a marker for regressive features in the hamster, appeared to be increased in long- term hypophysectomy as compared with short-day animals. Overall, these two conditions appeared to be similar.

A third objective of the present study was to determine if hypophysectomy in a seasonal breeder such as the hamster causes changes in the Sertoli cell similar to those produced by hypophysectomy in a nonseasonal breeder. It is clear that seasonal breeders are different from continuous breeders in their abilities to undergo periodic regressive changes and to reverse these changes. We wished to know if there were features of seasonal breeders and nonseasonal breeders that would cause them to respond differentially to hypophysectomy that would indicate a basic difference in their endocrine control. Remarkably similar changes were seen in hamster and rat Sertoli cells in response to hypophysectomy showing that rat and hamsters respond structurally to hypophysectomy and the subsequent depletion of hormones in a strictly comparable


manner. An interesting question is then “Are hamsters that have been hypophysectomized more capable of having their spermatogenesis restored by exogenous hormone administration than hypophysectomized rats, given that it is much more difficult to restore spermatogenesis in a rat testis regressed due to hypophysectomy (Sharpe, 1989) than one regressed in a seasonally breeding animal (Sinha Hikim et al., 1991; Bartke et al., 1979), i.e., one that is capable of restoring its own spermatogenesis during recrudescence (Matt and Stet- son, 1979; Nelson and Zucker, 1987).

At 6 days posthypophysectomy, considerably more extensive regressive features were seen in the hamster than in the rat. The percentage declines of various hormones in the hypophysectomized hamster resemble closely those in the hypophysectomized rat in the 6 day period. Plasma testosterone declined by 96% in the rat and 84% in the hamster; testicular testosterone declined 96% in the rat and 81% in the hamster; FSH declined 57% in the rat and 65% in the hamster. These percentage declines, however, do not necessarily re- flect the rate of decline, simply the level attained at 6 days after hypophysectomy. It is possible that either the rate of decline in the hamster was more rapid than in the rat or that the hamster is more sensitive to loss of hormonal stimulation.

Although not illustrated, the pattern of germ cell degeneration in the hamster was similar to that seen in the rat (Russell and Clermont, 1977; Russell et al., 1990; Ghosh et al., 1991) in that the initial degenera- tive features were seen in stage VII of the cycle and they involved preleptotene spermatocytes, pachytene spermatocytes, and step 7 and 19 spermatids. (In animals in which the testes were most severely regressed there was evidence that elongate spermatids in other stages were also undergoing degeneration). The ratio of germ cells to Sertoli cells revealed that in hamsters the step 7 spermatids appeared to be most sensitive to hormone depletion; whereas, in rats it was the pachytene spermatocytes that were the most sensitive. The preleptotene spermatocytes were least sensitive to hormone withdrawal in both conditions. At 6 days posthypophysectomy, there was clear histological evidence for degeneration of the four aforementioned germ cell types, however, the method of quantitation of viable germ cell types that was utilized was not sufficiently sensitive to reveal losses in these cell types. Thus the hamster can be added to the list of species which show stage VII sensitivity to hormonal withdrawal.

The present study also documents changes in FSH receptor concentration and content in hamsters after hypophysectomy. At 6 days posthypophysectomy, the concentration of FSH receptors as expressed per milligram of protein was not changed from the long-day animals but the content was significantly lower compared with long-day hamsters. These results agree with those found in the rat (Thanki and Steinberger, 1978; Tsutsui et al., 1985). Klemcke et al. (1987) however had reported an increase in the concentration of FSH receptors (per milligram protein) 6 days following hypophysectomy in the hamster. Tsutsui et al. (1985) reported a similar change in the mouse. In the rat (Ghosh et al., 1992), the FSH receptor concentration decreased between normal and long-term hypophysectomy, suggesting that a trend toward a decline should

be evident in periods between normal and long-term hypophysectomy. In the present study, a marked decrease in content also paralleled the study in the rat, indicating that receptors are lost. This is probably the case in the long-term hypophysectomized hamster although too little tissue was present to perform receptor assays.

In summary, the regressive features seen after short- term hypophysectomy are primarily in the interstitial compartment of the testis and in the diameter and length of the seminiferous tubule. Although many parameters were obtained for the Sertoli cell, only the cell surface areas were decreased after short-term hypophysectomy. Only after long-term hypophysectomy does the Sertoli cell manifest marked structural alter- ations. Compared with the Leydig cell, the Sertoli cell is a slow responder to loss of hormonal stimuli. The Sertoli cell in the seasonal breeding hamster responds to hormonal withdrawal brought about by hypophysectomy in a similar manner to hamsters undergoing photoperiodic regression except the quantitative regression is more severe after hypophysectomy. It appears that endocrine deprivation is the primary cause of both conditions since similar features are manifest. Hy- pophysectomy in the hamster and rat provide similar endocrine declines and quantitatively similar structural responses in the testis and specifically in the Ser- toli cell, although the hamster shows a more rapid decline in spermatogenesis. Overall, the data indicate that the Sertoli cell is sensitive to hormone depletion and shows a marked structural regression, but the structural decline of the Sertoli cell is not evident when germ cell degeneration is first prominent.

ACKNOWLEDGMENTS The assistance of Maureen Doran in sectioning,

Everett Woodard in printing, and Rebecca E. Cerven, Clare T. Fadden, and Juliana Gher in the hormone assays is greatly appreciated. We also acknowledge the gifts of the immunoreagents from the National Insti- tute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the National Hormone and Pituitary Program (NHPP), and from Dr. E. Talamantes, Uni- versity of California. This study was supported by NIH grant HD-20033 and NIH grant HD-13938 (L.E.R.).

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