THE AMERICAN JOURNAL OF ANATOMY 192:194-213 i 1991)
Reinitiation of Spermatogenesis by Exogenous Gonadotropins in a Seasonal Breeder, the Woodchuck (Marmota monax), During Gonadal Inactivity AMIYA P. SINHA HIKIM, INDRANI SINHA HIKIM, ARMAND0 G. AMADOR, ANDRZEJ BARTKE, ALAN WOOLF, AND LONNIE D. RUSSELL Cooperative Wildlife Research Laboratory (A.W., A.P.S.H.), Department of Zoology (A.W.);Department of Physiology (I.S.H., A.B., L.D.R.), Southern Illinois University, School of Medicine, Carbondale, Illinois 62901; and Department of Obstetrics and Gynecology, Southern Illinois University, School of Medicine, Springfield, Illinois 62794 (A.G.A.)
The present study was underINTRODUCTION ABSTRACT taken (1) to document structural and functional Despite considerable effort, the hormonal control of changes in the testes of seasonally breeding wood- mammalian spermatogenesis is not fully understood chuck during active and inactive states of sper- (see Steinberger and Steinberger, 1975; Ewing et al., matogenesis and (2) to evaluate the ability of 1980; Russell e t al., 1990). In continuous breeders, exogenous gonadotropins to reinitiate spermato- spermatogenesis has been maintained soon after horgenesis outside the breeding season. During sea- monal deprivation (Ahmad et al., 1975; Russell and sonal gonadal inactivity, there were significant Clermont, 1977; Bartlett et al., 1989) or reinitiated af(Pc0.05)reductions in volumes of several testicu- ter the period of complete testicular regression (Bocca1963; Vernon et al., 1975; Huang et al., 1987) lar features (testis, seminiferous tubules, tubular bella, following exogenous administration of hormone(s). In lumen, interstitial tissue, individual Leydig cells, seasonal breeders, spermatogenesis is naturally reiniLeydig cell nuclei, and Leydig cell cytoplasm) as tiated in animals that have undergone annual gonadal compared with gonadally active animals. The di- regression (see Bartke, 1985). Results obtained from ameter of the seminiferous tubules was decreased continuous breeders have indicated t h a t testosterone by 26%, and Leydig cell numbers also declined in (T)alone can quantitatively restore spermatogenesis in the regressed testes. These changes were accom- rats made azoospermic with ethane dimethane sulpanied by a decline in testosterone (T) levels in fonate (Sharpe et al., 1990) or by active immunization both plasma and testis, and reduction in epithelial against luteinizing hormone or gonadotropin-releasing height of accessory reproductive organs. A hor- hormone (Awoniyi et al., 1989). Seasonally breeding mammals are good models in monal regimen was developed that would reini- which to study hormonal requirements for reinitiation tiate spermatogenesis in captive, sexually quies- of spermatogenesis. The seasonally breeding mammals cent woodchucks. A combination of PMSG and with their annual changes in spermatogenic activity hCG markedly stimulated testicular growth and provide a physiological system (Sinha Hikim et al., function and restored spermatogenesis qualita- 1989a,b) for studying the hormonal control of spertively. Quantitatively normal spermatogenesis matogenesis without complications which may result was restored in 2 of 6 treated males. Morphometric from unnatural experimental manipulation such as hyanalyses revealed substantial increases in semi- pophysectomy. However, information on hormonal inniferous tubular diameter and in the volume of duction of reinitiation of spermatogenesis in seasonal seminiferous tubules, tubular lumen, total Leydig breeders outside their breeding season is limited. Bartke et al. (1979) reported restoration of fertility in shortcells, and individual Leydig cells in the hormone- photoperiod-induced, sterile, male golden hamsters treated animals. These increased values corre- with a single ectopic pituitary homograft from a n sponded to 99,75,68,51, and 200%,respectively, of adult, gonadally active female. Recently, Sundqvist et the values measured in naturally active wood- al. (1989) indicated the possibility of inducing and/or chucks. Leydig cell numbers, however, remained maintaining testicular function in another seasonally unchanged and approximated only 31% of the breeding rodent, the woodchuck, outside its normally number found in naturally active testes. Hormonal brief breeding season by a simple regimen of PMSG or stimulation also resulted in a significant rise in PMSG + hCG injections. To our knowledge, there have serum T as well as in the total content of testicular T, and a marked increase in epithelial height in various accessory reproductive glands. The most effective hormonal protocol for stimulating sperReceived February 6, 1991. Accepted May 17, 1991. matogenesis was treatment with 12.5 IU of PMSG Address reprint requests to Dr. Lonnie D. Russell, Laboratory of twice a week for 4 weeks followed by 12.5 IU of Structural Biology, Department of Physiology, School of Medicine, PMSG + 25 IU of hCG twice a week for 4 weeks. Southern Illinois University, Carbondale, IL 62901. Q 1991 WILEY-LISS.
INC.
REINITIATION OF SPERMATOGENESIS IN T H E WOODCHUCK
been no studies in any seasonally breeding mammalian species in which quantitative restoration of spermatogenesis outside of their normal breeding season was achieved with exogenously administered hormone(s). The woodchuck is a seasonal breeder with a marked annual testicular cycle (Rasmussen, 1918; Christian et al., 1972; Baldwin et al., 1985). It has been used extensively as a model for studying several human conditions including obesity, vascular diseases, and, most importantly, viral hepatitis and hepatocellular carcinoma (see Young and Simms, 1979; Snyder, 1985). Because of the potential importance of this species in these areas of research, induction of fertility outside of the normal breeding season would be of practical significance to develop and maintain a colony for biomedical studies. The present study was designed to develop a hormonal regimen for the reinitiation of spermatogenesis in captive, sexually quiescent woodchucks. Using a variety of objective and subjective criteria, we have characterized structural changes in the testis and in the accessory reproductive glands in this species during the various states of gonadal activity and have evaluated the degree of restoration of spermatogenesis following hormone treatments. MATERIALS AND METHODS Animals
Thirty-eight adult (more than 2 years old) male woodchucks (Marmota moncwc), weighing between 3.9 and 5.0 kg, were used in this study. Animals were either live trapped in the vicinity of Carbondale (Jackson County, IL) or obtained from Cornell University (Ithaca, NY). They were housed individually in rabbit cages (0.61m long x 0.60 m wide x 0.49 m high) with woodchips or straw as nesting materials. Temperature was maintained a t 22 2 2°C and photoperiod a t 12 h r of light and 12 h r of darkness. Rabbit chow (Teklad, Harlan Sprague-Dawley Inc., Co., Madison, WI), supplemented with fresh vegetables and/or fruits 2-3 times/ week, and water were provided ad libitum. Protocol
Experiment #1
Objectives of the first experiment were (1)to assess, both qualitatively and quantitatively, spermatogenesis and various testicular measurements as well as accessory reproductive glands in gonadally active, gonadal regressed, and hormone-treated animals; and (2) to determine how closely the hormone-treated woodchucks resembled naturally gonadally active animals. Three groups of randomly selected male woodchucks, naturally active ( n= 61, gonadally regressed ( n= 51, and hormone-treated ( n= 6) were utilized. Animals in the naturally active group were sacrificed during their natural breeding season (February and March) when spermatogenic activity is maximal (Christian et al., 1972; Baldwin et al., 1985). Hormone treatment began in mid-October, when the animals were still sexually regressed; there was no scrota1 development and it was not possible to palpate the abdominal testes. Males in the hormone-treated group received subcutaneous injections of 25 IU of pregnant mare serum gonadotropin (PMSG) twice a week for 4 weeks, followed by twice weekly injections of :!5 IU of PMSG + 50 IU of human chorionic gonado-
195
tropin (hCG) (CALBIOCHEM, Behring Diagnostics, La Jolla, CA) for a n additional 4 weeks. The animals in the regressed group were given equal volumes of vehicle (0.9% saline) over the 8-week periods. Animals in the regressed and hormone-treated groups were euthanized 1 day after the last injection by intravenous injection of a commercial euthanasia solution (26% sodium pentobarbital) after inducing anesthesia with a mixture of ketamine (Ketaset, Aveco Co., Fort Dodge, IA) and xylazine (Rompun, Haver, Mobay Corp., KS). Blood samples were collected from each animal by cardiac puncture, and serum was prepared and stored frozen a t -20°C for subsequent determination of testosterone (TI. One testis per woodchuck was removed and weighed, and its volume was measured by water displacement (Steer, 1981).The specific gravity of the testis was determined by dividing testicular weight by its volume (Steer, 1981). Testicular tissue was stored frozen a t 70°C for subsequent determination of steroid levels. The contralateral testis from each woodchuck was fixed by vascular perfusion through the testicular artery with 5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) preceded by a brief saline wash. Regressed testes were often difficult to perfuse by this technique, so if perfusion failed they were fixed by immersion. Epididymides were dissected out and the caudal portion of one epididymis was used to determine the number of epididymal spermatozoa by the homogenization technique (see below). The contralatera1 epididymis from each animal was used for histological study. To examine the morphological changes in male accessory reproductive organs, the entire reproductive tracts from two randomly selected woodchucks in each group were carefully dissected out and fixed in 10% neutral-buffered formalin. Tissues taken for subsequent histological analyses included prostate, seminal vesicle, and Cowper’s gland. ~
Experiment #2
The objectives of the second experiment (designed after the results of the first experiment were known) were (1)to determine if motile sperm were present in the epididymis following hormone treatment, (2) to select doses of gonadotropins for optimal stimulation of testicular activity, (3) to determine whether pretreatment with follicle-stimulating hormone (FSH) instead of PMSG would enhance the efficacy of our hormone treatment protocol, and (4)to determine whether exogenously administered gonadotropins can reinitiate spermatogenesis when the treatment is started a t the time of maximal testicular regression. Experiments were started in July, when testicular involution is maximal (Christian et al., 1972; Baldwin et al., 1985). Twenty-one sexually regressed woodchucks were divided into 4 groups to receive one of the following treatments: twice weekly injections of saline for 8 weeks (regressed; n = 4);3 IU of FSH daily for 4 weeks then biweekly injections of 12.5 IU of PMSG + 25 IU of hCG for additional 4 weeks (FSH + PMSGhCG group; n = 5); twice weekly injection of 12.5 IU of PMSG for 4 weeks, followed by biweekly injection of 12.5 IU of PMSG + 25 IU of hCG for another 4-week period (low PMSG-hCG group; n = 6); and repetition of the hormonal regimen used in experiment #I (high PMSG-hCG group; n = 6). All animals were euthanized
A.P. SINHA HIKIM ET AL.
196
1 day after the last injection and specimens were collected as described for Experiment # 1. In order to evaluate sperm motility after hormone treatment, a portion of cauda epididymides from each of the hormonetreated woodchucks was minced in a few drops of normal saline on a glass slide and the suspension was examined with the light microscope. Specimen Preparation for Light and Electron Microscopic Studies
After glutaraldehyde fixation, testis tissue was diced into small pieces (1x 2 x 2 mm) and postfixed with 1% osmium and 1.25% potassium ferrocyanide mixture (Russell and Burguet, 1977), dehydrated in a graded series of ethanol, and embedded in Araldite (CY212). During embedding, tissue blocks were oriented so that seminiferous tubules could be sectioned transversely yielding round or rounded tubular profiles. Embedded tissue blocks were then sectioned with a Sorval MT-1 ultramicrotome a t 0.92 km (Sinha Hikim et al., 1988b), and stained with 1%toluidine blue for histological observations and for routine morphometric analyses of testicular components a t the light-microscope level. For electron microscopic studies, thin sections from the randomly selected tissue blocks showing a silver-gold interference color were cut with a Reichert ultramicrotome and stained with uranyl acetate and lead citrate and examined on a Hitachi H500H electron microscope. Representative samples of accessory reproductive glands from each group of woodchucks were embedded in paraffin, sectioned a t 5 km, stained with hematoxylin and eosin, and mounted. Morphometric Procedures Volume density determinations
Toluidine blue-stained thick sections were examined with a Microphot-FX microscope (Nikon, Garden City, NY) equipped with a bright-field condenser. The volume density of testicular components (including seminiferous tubules, tubular lumina, interstitium, and Leydig cells) was obtained by point counting (Weibel, 1979; Sinha Hikim et al., 1988b). Ten randomly selected sections per animal in each group were examined with a 4 0 objective ~ and a 1 0 eyepiece, ~ the latter fitted with a square lattice containing 441 intersections. The number of intersections (“hits”)on pertinent structures over the entire tissue section was counted by predetermined and systematic movement of sections across the grid without overlap. Volume density (V,) of each testicular component (volume of a given testicular component per unit volume of testis) was obtained by dividing the sum of points falling on each structure by the total number of points over the tissue. The results were expressed as a percentage of the testis volume (V,%) obtained by multiplying the volume density of each testis component by 100. The absolute volume (V) of each of the testis components was then determined by multiplying its volume density by fresh testis volume. A similar approach employing the same stereologic principle was used to obtain the volumetric composition of the interstitial tissue (V,) in each group of woodchucks.
Seminiferous tubule length and diameter
The diameters of 20 randomly selected transverse sections of seminiferous tubules were measured for each animal across the minor axis of their cross-sectioned profiles (Wing and Christensen, 1982) with a n ocular micrometer calibrated by means of a stage micrometer, and a 10 x objective lens. The total length of the seminiferous tubule (L) was calculated by the standard equation ( L= V,JIT?) for a tube model (used by Wing and Christensen, 1982), where V,, is the tubular volume per testis a s measured above and r is the mean radius as determined from the tubular diameter. Leydig cell volume and numerical density
Measurements for the determination of the Leydig cell volume were carried out a t 1,000 x magnification. Since the Leydig cell nuclei are spherical in woodchucks, the volume of a n individual nucleus was calculated from the mean diameter of the nucleus using the equation V, = 116rD3 (Steer, 1981).The mean diameter of the nucleus (D)was obtained by direct measurement of its cross-sectioned profiles in serial sections. Fifty nuclear profiles were measured per animal in each group. The volume of a n average Leydig cell (V,) was derived from its nuclear volume (V,) a s determined above and the volume density of the nucleus W,,) within the cell using the equation V, = V,/V,,, (Williams, 1977; Bolender, 1979). The volume density of the Leydig cell nucleus (V,,) within the cell was obtained by dividing the number of points falling on the nuclei by the total number of points lying over the Leydig cells (nucleus and cytoplasm), using the same ocular grid and the testicular sections used to calculate Vv% of the various testicular components. The cytoplasmic volume ( V J was then calculated by subtracting V, from V,. The numerical density of the Leydig cell (N,) was obtained by dividing the volume density of the Leydig cells by the volume of a n individual Leydig cell (Kaler and Neaves, 1978; Sinha Hikim et al., 198813). The absolute number of Leydig cells per testis was determined by multiplying the number of Leydig cells per unit volume of the testis by the fresh testicular volume. Sperm count
Epididymal sperm reserves was determined by hemocytometric counting of epididymal homogenates as described previously (Amann and Lambiase, 1968). One epididymis from each woodchuck was freed from surrounding tissues, cut into small pieces, and homogenized in 2.5 ml of 0.9% NaCl solution containing 0.05% Triton-X 100 (viv) for 15 sec with Super Dispax Tissumizer (Model SDT-182). Without further dilution of the suspension, the number of sperm in homogenates were counted using a hemocytometer. Epithelial height of accessory reproductive glands
Epithelial heights (principal cells of the epididymis) were determined in 1-pm plastic sections using a n ocular micrometer fitted to the eyepiece. For sex accessory organs (including prostate, seminal vesicle, and Cowper’s gland), measurements were obtained from 5pm paraffin sections stained with hematoxylin and eosin. Twenty-five randomly chosen sites were mea-
REINITIATION OF SPERMATOGENESIS IN THE WOODCHUCK
197
TABLE 1. Body weight and testis weight, volume, and specific gravity (means in woodchucks used in Experiment #1'
Body weight (kg) Testis weight (gm) Testis volume (ml) Specific gravity
Regressed (N=5) 3.86 O.3la2 1.23 2 0.08" 1.10 t 0.13' 1.04 t 0.004"
Active (N=6) 3.95 k 0.23" 3.75 t 0.42" 3.63 ?C 0.39" 1.03 2 0.006"
*
&
SE)
Hormone-treated (N=6) 4.17 2 0.18" 2.41 2 0.25' 2.32 2 0.25h 1.04 2 0.002"
'Gonadally regressed animals received subcutaneous injections of 25 IU PMSG twice a week for 4 weeks, followed by twice weekly injections of 25 IU of PMSG + 50 IU of hCG for a n additional 4 weeks. 'In each row, means with unlike superscripts are significantly (P
Reinitiation of Spermatogenesis by Exogenous Gonadotropins in a Seasonal Breeder, the Woodchuck (Marmota monax), During Gonadal Inactivity AMIYA P. SINHA HIKIM, INDRANI SINHA HIKIM, ARMAND0 G. AMADOR, ANDRZEJ BARTKE, ALAN WOOLF, AND LONNIE D. RUSSELL Cooperative Wildlife Research Laboratory (A.W., A.P.S.H.), Department of Zoology (A.W.);Department of Physiology (I.S.H., A.B., L.D.R.), Southern Illinois University, School of Medicine, Carbondale, Illinois 62901; and Department of Obstetrics and Gynecology, Southern Illinois University, School of Medicine, Springfield, Illinois 62794 (A.G.A.)
The present study was underINTRODUCTION ABSTRACT taken (1) to document structural and functional Despite considerable effort, the hormonal control of changes in the testes of seasonally breeding wood- mammalian spermatogenesis is not fully understood chuck during active and inactive states of sper- (see Steinberger and Steinberger, 1975; Ewing et al., matogenesis and (2) to evaluate the ability of 1980; Russell e t al., 1990). In continuous breeders, exogenous gonadotropins to reinitiate spermato- spermatogenesis has been maintained soon after horgenesis outside the breeding season. During sea- monal deprivation (Ahmad et al., 1975; Russell and sonal gonadal inactivity, there were significant Clermont, 1977; Bartlett et al., 1989) or reinitiated af(Pc0.05)reductions in volumes of several testicu- ter the period of complete testicular regression (Bocca1963; Vernon et al., 1975; Huang et al., 1987) lar features (testis, seminiferous tubules, tubular bella, following exogenous administration of hormone(s). In lumen, interstitial tissue, individual Leydig cells, seasonal breeders, spermatogenesis is naturally reiniLeydig cell nuclei, and Leydig cell cytoplasm) as tiated in animals that have undergone annual gonadal compared with gonadally active animals. The di- regression (see Bartke, 1985). Results obtained from ameter of the seminiferous tubules was decreased continuous breeders have indicated t h a t testosterone by 26%, and Leydig cell numbers also declined in (T)alone can quantitatively restore spermatogenesis in the regressed testes. These changes were accom- rats made azoospermic with ethane dimethane sulpanied by a decline in testosterone (T) levels in fonate (Sharpe et al., 1990) or by active immunization both plasma and testis, and reduction in epithelial against luteinizing hormone or gonadotropin-releasing height of accessory reproductive organs. A hor- hormone (Awoniyi et al., 1989). Seasonally breeding mammals are good models in monal regimen was developed that would reini- which to study hormonal requirements for reinitiation tiate spermatogenesis in captive, sexually quies- of spermatogenesis. The seasonally breeding mammals cent woodchucks. A combination of PMSG and with their annual changes in spermatogenic activity hCG markedly stimulated testicular growth and provide a physiological system (Sinha Hikim et al., function and restored spermatogenesis qualita- 1989a,b) for studying the hormonal control of spertively. Quantitatively normal spermatogenesis matogenesis without complications which may result was restored in 2 of 6 treated males. Morphometric from unnatural experimental manipulation such as hyanalyses revealed substantial increases in semi- pophysectomy. However, information on hormonal inniferous tubular diameter and in the volume of duction of reinitiation of spermatogenesis in seasonal seminiferous tubules, tubular lumen, total Leydig breeders outside their breeding season is limited. Bartke et al. (1979) reported restoration of fertility in shortcells, and individual Leydig cells in the hormone- photoperiod-induced, sterile, male golden hamsters treated animals. These increased values corre- with a single ectopic pituitary homograft from a n sponded to 99,75,68,51, and 200%,respectively, of adult, gonadally active female. Recently, Sundqvist et the values measured in naturally active wood- al. (1989) indicated the possibility of inducing and/or chucks. Leydig cell numbers, however, remained maintaining testicular function in another seasonally unchanged and approximated only 31% of the breeding rodent, the woodchuck, outside its normally number found in naturally active testes. Hormonal brief breeding season by a simple regimen of PMSG or stimulation also resulted in a significant rise in PMSG + hCG injections. To our knowledge, there have serum T as well as in the total content of testicular T, and a marked increase in epithelial height in various accessory reproductive glands. The most effective hormonal protocol for stimulating sperReceived February 6, 1991. Accepted May 17, 1991. matogenesis was treatment with 12.5 IU of PMSG Address reprint requests to Dr. Lonnie D. Russell, Laboratory of twice a week for 4 weeks followed by 12.5 IU of Structural Biology, Department of Physiology, School of Medicine, PMSG + 25 IU of hCG twice a week for 4 weeks. Southern Illinois University, Carbondale, IL 62901. Q 1991 WILEY-LISS.
INC.
REINITIATION OF SPERMATOGENESIS IN T H E WOODCHUCK
been no studies in any seasonally breeding mammalian species in which quantitative restoration of spermatogenesis outside of their normal breeding season was achieved with exogenously administered hormone(s). The woodchuck is a seasonal breeder with a marked annual testicular cycle (Rasmussen, 1918; Christian et al., 1972; Baldwin et al., 1985). It has been used extensively as a model for studying several human conditions including obesity, vascular diseases, and, most importantly, viral hepatitis and hepatocellular carcinoma (see Young and Simms, 1979; Snyder, 1985). Because of the potential importance of this species in these areas of research, induction of fertility outside of the normal breeding season would be of practical significance to develop and maintain a colony for biomedical studies. The present study was designed to develop a hormonal regimen for the reinitiation of spermatogenesis in captive, sexually quiescent woodchucks. Using a variety of objective and subjective criteria, we have characterized structural changes in the testis and in the accessory reproductive glands in this species during the various states of gonadal activity and have evaluated the degree of restoration of spermatogenesis following hormone treatments. MATERIALS AND METHODS Animals
Thirty-eight adult (more than 2 years old) male woodchucks (Marmota moncwc), weighing between 3.9 and 5.0 kg, were used in this study. Animals were either live trapped in the vicinity of Carbondale (Jackson County, IL) or obtained from Cornell University (Ithaca, NY). They were housed individually in rabbit cages (0.61m long x 0.60 m wide x 0.49 m high) with woodchips or straw as nesting materials. Temperature was maintained a t 22 2 2°C and photoperiod a t 12 h r of light and 12 h r of darkness. Rabbit chow (Teklad, Harlan Sprague-Dawley Inc., Co., Madison, WI), supplemented with fresh vegetables and/or fruits 2-3 times/ week, and water were provided ad libitum. Protocol
Experiment #1
Objectives of the first experiment were (1)to assess, both qualitatively and quantitatively, spermatogenesis and various testicular measurements as well as accessory reproductive glands in gonadally active, gonadal regressed, and hormone-treated animals; and (2) to determine how closely the hormone-treated woodchucks resembled naturally gonadally active animals. Three groups of randomly selected male woodchucks, naturally active ( n= 61, gonadally regressed ( n= 51, and hormone-treated ( n= 6) were utilized. Animals in the naturally active group were sacrificed during their natural breeding season (February and March) when spermatogenic activity is maximal (Christian et al., 1972; Baldwin et al., 1985). Hormone treatment began in mid-October, when the animals were still sexually regressed; there was no scrota1 development and it was not possible to palpate the abdominal testes. Males in the hormone-treated group received subcutaneous injections of 25 IU of pregnant mare serum gonadotropin (PMSG) twice a week for 4 weeks, followed by twice weekly injections of :!5 IU of PMSG + 50 IU of human chorionic gonado-
195
tropin (hCG) (CALBIOCHEM, Behring Diagnostics, La Jolla, CA) for a n additional 4 weeks. The animals in the regressed group were given equal volumes of vehicle (0.9% saline) over the 8-week periods. Animals in the regressed and hormone-treated groups were euthanized 1 day after the last injection by intravenous injection of a commercial euthanasia solution (26% sodium pentobarbital) after inducing anesthesia with a mixture of ketamine (Ketaset, Aveco Co., Fort Dodge, IA) and xylazine (Rompun, Haver, Mobay Corp., KS). Blood samples were collected from each animal by cardiac puncture, and serum was prepared and stored frozen a t -20°C for subsequent determination of testosterone (TI. One testis per woodchuck was removed and weighed, and its volume was measured by water displacement (Steer, 1981).The specific gravity of the testis was determined by dividing testicular weight by its volume (Steer, 1981). Testicular tissue was stored frozen a t 70°C for subsequent determination of steroid levels. The contralateral testis from each woodchuck was fixed by vascular perfusion through the testicular artery with 5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) preceded by a brief saline wash. Regressed testes were often difficult to perfuse by this technique, so if perfusion failed they were fixed by immersion. Epididymides were dissected out and the caudal portion of one epididymis was used to determine the number of epididymal spermatozoa by the homogenization technique (see below). The contralatera1 epididymis from each animal was used for histological study. To examine the morphological changes in male accessory reproductive organs, the entire reproductive tracts from two randomly selected woodchucks in each group were carefully dissected out and fixed in 10% neutral-buffered formalin. Tissues taken for subsequent histological analyses included prostate, seminal vesicle, and Cowper’s gland. ~
Experiment #2
The objectives of the second experiment (designed after the results of the first experiment were known) were (1)to determine if motile sperm were present in the epididymis following hormone treatment, (2) to select doses of gonadotropins for optimal stimulation of testicular activity, (3) to determine whether pretreatment with follicle-stimulating hormone (FSH) instead of PMSG would enhance the efficacy of our hormone treatment protocol, and (4)to determine whether exogenously administered gonadotropins can reinitiate spermatogenesis when the treatment is started a t the time of maximal testicular regression. Experiments were started in July, when testicular involution is maximal (Christian et al., 1972; Baldwin et al., 1985). Twenty-one sexually regressed woodchucks were divided into 4 groups to receive one of the following treatments: twice weekly injections of saline for 8 weeks (regressed; n = 4);3 IU of FSH daily for 4 weeks then biweekly injections of 12.5 IU of PMSG + 25 IU of hCG for additional 4 weeks (FSH + PMSGhCG group; n = 5); twice weekly injection of 12.5 IU of PMSG for 4 weeks, followed by biweekly injection of 12.5 IU of PMSG + 25 IU of hCG for another 4-week period (low PMSG-hCG group; n = 6); and repetition of the hormonal regimen used in experiment #I (high PMSG-hCG group; n = 6). All animals were euthanized
A.P. SINHA HIKIM ET AL.
196
1 day after the last injection and specimens were collected as described for Experiment # 1. In order to evaluate sperm motility after hormone treatment, a portion of cauda epididymides from each of the hormonetreated woodchucks was minced in a few drops of normal saline on a glass slide and the suspension was examined with the light microscope. Specimen Preparation for Light and Electron Microscopic Studies
After glutaraldehyde fixation, testis tissue was diced into small pieces (1x 2 x 2 mm) and postfixed with 1% osmium and 1.25% potassium ferrocyanide mixture (Russell and Burguet, 1977), dehydrated in a graded series of ethanol, and embedded in Araldite (CY212). During embedding, tissue blocks were oriented so that seminiferous tubules could be sectioned transversely yielding round or rounded tubular profiles. Embedded tissue blocks were then sectioned with a Sorval MT-1 ultramicrotome a t 0.92 km (Sinha Hikim et al., 1988b), and stained with 1%toluidine blue for histological observations and for routine morphometric analyses of testicular components a t the light-microscope level. For electron microscopic studies, thin sections from the randomly selected tissue blocks showing a silver-gold interference color were cut with a Reichert ultramicrotome and stained with uranyl acetate and lead citrate and examined on a Hitachi H500H electron microscope. Representative samples of accessory reproductive glands from each group of woodchucks were embedded in paraffin, sectioned a t 5 km, stained with hematoxylin and eosin, and mounted. Morphometric Procedures Volume density determinations
Toluidine blue-stained thick sections were examined with a Microphot-FX microscope (Nikon, Garden City, NY) equipped with a bright-field condenser. The volume density of testicular components (including seminiferous tubules, tubular lumina, interstitium, and Leydig cells) was obtained by point counting (Weibel, 1979; Sinha Hikim et al., 1988b). Ten randomly selected sections per animal in each group were examined with a 4 0 objective ~ and a 1 0 eyepiece, ~ the latter fitted with a square lattice containing 441 intersections. The number of intersections (“hits”)on pertinent structures over the entire tissue section was counted by predetermined and systematic movement of sections across the grid without overlap. Volume density (V,) of each testicular component (volume of a given testicular component per unit volume of testis) was obtained by dividing the sum of points falling on each structure by the total number of points over the tissue. The results were expressed as a percentage of the testis volume (V,%) obtained by multiplying the volume density of each testis component by 100. The absolute volume (V) of each of the testis components was then determined by multiplying its volume density by fresh testis volume. A similar approach employing the same stereologic principle was used to obtain the volumetric composition of the interstitial tissue (V,) in each group of woodchucks.
Seminiferous tubule length and diameter
The diameters of 20 randomly selected transverse sections of seminiferous tubules were measured for each animal across the minor axis of their cross-sectioned profiles (Wing and Christensen, 1982) with a n ocular micrometer calibrated by means of a stage micrometer, and a 10 x objective lens. The total length of the seminiferous tubule (L) was calculated by the standard equation ( L= V,JIT?) for a tube model (used by Wing and Christensen, 1982), where V,, is the tubular volume per testis a s measured above and r is the mean radius as determined from the tubular diameter. Leydig cell volume and numerical density
Measurements for the determination of the Leydig cell volume were carried out a t 1,000 x magnification. Since the Leydig cell nuclei are spherical in woodchucks, the volume of a n individual nucleus was calculated from the mean diameter of the nucleus using the equation V, = 116rD3 (Steer, 1981).The mean diameter of the nucleus (D)was obtained by direct measurement of its cross-sectioned profiles in serial sections. Fifty nuclear profiles were measured per animal in each group. The volume of a n average Leydig cell (V,) was derived from its nuclear volume (V,) a s determined above and the volume density of the nucleus W,,) within the cell using the equation V, = V,/V,,, (Williams, 1977; Bolender, 1979). The volume density of the Leydig cell nucleus (V,,) within the cell was obtained by dividing the number of points falling on the nuclei by the total number of points lying over the Leydig cells (nucleus and cytoplasm), using the same ocular grid and the testicular sections used to calculate Vv% of the various testicular components. The cytoplasmic volume ( V J was then calculated by subtracting V, from V,. The numerical density of the Leydig cell (N,) was obtained by dividing the volume density of the Leydig cells by the volume of a n individual Leydig cell (Kaler and Neaves, 1978; Sinha Hikim et al., 198813). The absolute number of Leydig cells per testis was determined by multiplying the number of Leydig cells per unit volume of the testis by the fresh testicular volume. Sperm count
Epididymal sperm reserves was determined by hemocytometric counting of epididymal homogenates as described previously (Amann and Lambiase, 1968). One epididymis from each woodchuck was freed from surrounding tissues, cut into small pieces, and homogenized in 2.5 ml of 0.9% NaCl solution containing 0.05% Triton-X 100 (viv) for 15 sec with Super Dispax Tissumizer (Model SDT-182). Without further dilution of the suspension, the number of sperm in homogenates were counted using a hemocytometer. Epithelial height of accessory reproductive glands
Epithelial heights (principal cells of the epididymis) were determined in 1-pm plastic sections using a n ocular micrometer fitted to the eyepiece. For sex accessory organs (including prostate, seminal vesicle, and Cowper’s gland), measurements were obtained from 5pm paraffin sections stained with hematoxylin and eosin. Twenty-five randomly chosen sites were mea-
REINITIATION OF SPERMATOGENESIS IN THE WOODCHUCK
197
TABLE 1. Body weight and testis weight, volume, and specific gravity (means in woodchucks used in Experiment #1'
Body weight (kg) Testis weight (gm) Testis volume (ml) Specific gravity
Regressed (N=5) 3.86 O.3la2 1.23 2 0.08" 1.10 t 0.13' 1.04 t 0.004"
Active (N=6) 3.95 k 0.23" 3.75 t 0.42" 3.63 ?C 0.39" 1.03 2 0.006"
*
&
SE)
Hormone-treated (N=6) 4.17 2 0.18" 2.41 2 0.25' 2.32 2 0.25h 1.04 2 0.002"
'Gonadally regressed animals received subcutaneous injections of 25 IU PMSG twice a week for 4 weeks, followed by twice weekly injections of 25 IU of PMSG + 50 IU of hCG for a n additional 4 weeks. 'In each row, means with unlike superscripts are significantly (P
