I
ANDROLOGIA
24, 349-354 (1992)
ACCEPTED: APRIL4, 1992
Morphometric and ultrastructural studies on the rat testis following administration of antiserum to human seminal plasma inhibin K. Gopalkrishnan, G. Vanage and A. R. Sheth Key words. Testis - morphology - seminal plasma inhibin - rat.
Summary. A study was undertaken to see the effects of antiserum to human seminal plasma inhibin (hSPI) on the morphology of rat testis. Morphometric, light microscopic, and ultrastructural studies were done on rat testis after 4, 8, and 12 weeks of administration of antiserum to hSPI. Daily sperm production rate was also estimated by histometric method. The light microscopic analysis showed a slight decrease in tubular diameter which was not significant. T h e degenerative changes in the tubules were marked after 12 weeks of treatment. The daily sperm production rate was reduced by 50% after 12 weeks of treatment. The ultrastructural study revealed phagocytosis of elongated spermatids and spermatozoa enclosed in a vacuole surrounded by Sertoli cells. The Sertoli cells were dedifferentiated into an immature type. The spermatogonia were not affected. The treatment with antiserum to hSPI alters testicular morphology at the spermatid and mature spermatozoa level. Since treatment with AshSPI is known to elevate the FSH level it appears that the morphological changes correlate with the endocrine status.
Introduction Inhibin isolated from human seminal plasma (Thakur et al., 1981; Sheth et al., 1984) has been shown to function similarly to sperm coating antigens (Johanson et al., 1984). Antiserum to this human seminal plasma inhibin (AshSPI) was shown to cause sperm agglutination and impairInstitute for Research in Reproduction (ICMR), Parel, Bombay, India. Correspondence: Dr A . R. Sheth, Director, Institute for Research in Reproduction, Jehangir Merwanji Street, Parel, Bombay 400 012, India.
ment of cervical mucus penetration and spermegg attachment (Bandivdekar et al., 1987). Previous studies have shown that inhibin has an importance in the physiological function of spermatozoa. The present paper describes the morphometric studies on rat testis after treatment with AshSPI for different durations. The paper also describes the ultrastructural features of the Sertoli cells of normal testis and compares the findings with the fine structure of the Sertoli cells from testes of treated rats.
Materials and methods Preparation of human seminal plasma inhibin (HSPI) The antigen HSPI was purified according to the procedure of Thakur et al. ( 1981) . This homogenous peptide was used to raise the highly specific antibodies by active immunization. The checks for specific antisera were carried out as reported earlier (Shanbaug et al., 1985). These antibodies were used in this study. Adult male rats of Holtzman strain (75 d old; 250-300 g body weight) were subcutaneously injected biweekly with 0.1 ml of either AshSPI or normal rabbit serum (NRS) for 4, 8, and 12 weeks. Animals were killed 2 d after the last injection. The testes were removed and small pieces fixed in modified Karnovsky’s fluid (David et al., 1973), post-fixed in 1% OSO, processed further and embedded in araldite. Semithin sections (0.5 pm) were stained with toluidine blue and observed under bright field optics at a magnification of 400 x and 1000 x . A differential count of cell types was obtained using Chalkley’s point sampling method (1943). Precision was obtained by counting 1000 points over one or more seminiferous tubules. Tubular diameter was measured with an occular micrometer based on 25 tubules/
350
K. GOPALKRISHNAN ET A L .
testis. Care was taken that seminiferous tubules cross sections evaluated, are localized at distant sites in the testis so that they do not represent the same area. Daily sperm production (DSP) For histometric analysis individual seminiferous tubules were separated under a transillumination stereomicroscope at a magnification of 40- 150 x . Tubules measuring 1-2 mm length were dissected, fixed, processed and embedded in araldite. Estimation of DSP was calculated as described earlier (Gopalkrishnan et al., 1987). Hormone estimations Serum FSH and LH were estimated by radioimmunoassay (RIA) using rFSH and rLH kits supplied by NIDDK, M D USA. Electron microscopy Testicular tissue obtained from control (NRS treated) and treated rats were fixed in Karnovsky's fluid for 1 h and postfixed in lo/, OSO, ( 1 h) and after dehydration of samples in acetone the samples were embedded in araldite. Ultrathin sections were cut on a Reichert Ultracut microtome, contrasted with uranyl acetate and lead citrate according to the method of Reynolds (1963). These sections were observed under a Philips 400 EM transmission electron microscope at 80 KV.
Results Morphometry During the present investigation no changes in the testicular weights were observed after different durations of treatment. Daily sperm production as judged by morphometric analysis was reduced b; 50% (Table 1).
Table 1. Tubular diameter and daily sperm production rate in control and in antiserum-treated adult male rats
Table 2. Effect of biweekly administration of 0.1 ml of AshSPI or NRS on circulatory levels of FSH, LH and Prolactin (Prl) in adult male rats (Mean? SE) Hormones
NRS (ng rn1-l)
FSH LH Prl
16k0.3 26k 1.6 204 14.3
AshSPI (ng m1-l) 47+4.1*
25 2.4 205+ 18.4
+
*P
ANDROLOGIA
24, 349-354 (1992)
ACCEPTED: APRIL4, 1992
Morphometric and ultrastructural studies on the rat testis following administration of antiserum to human seminal plasma inhibin K. Gopalkrishnan, G. Vanage and A. R. Sheth Key words. Testis - morphology - seminal plasma inhibin - rat.
Summary. A study was undertaken to see the effects of antiserum to human seminal plasma inhibin (hSPI) on the morphology of rat testis. Morphometric, light microscopic, and ultrastructural studies were done on rat testis after 4, 8, and 12 weeks of administration of antiserum to hSPI. Daily sperm production rate was also estimated by histometric method. The light microscopic analysis showed a slight decrease in tubular diameter which was not significant. T h e degenerative changes in the tubules were marked after 12 weeks of treatment. The daily sperm production rate was reduced by 50% after 12 weeks of treatment. The ultrastructural study revealed phagocytosis of elongated spermatids and spermatozoa enclosed in a vacuole surrounded by Sertoli cells. The Sertoli cells were dedifferentiated into an immature type. The spermatogonia were not affected. The treatment with antiserum to hSPI alters testicular morphology at the spermatid and mature spermatozoa level. Since treatment with AshSPI is known to elevate the FSH level it appears that the morphological changes correlate with the endocrine status.
Introduction Inhibin isolated from human seminal plasma (Thakur et al., 1981; Sheth et al., 1984) has been shown to function similarly to sperm coating antigens (Johanson et al., 1984). Antiserum to this human seminal plasma inhibin (AshSPI) was shown to cause sperm agglutination and impairInstitute for Research in Reproduction (ICMR), Parel, Bombay, India. Correspondence: Dr A . R. Sheth, Director, Institute for Research in Reproduction, Jehangir Merwanji Street, Parel, Bombay 400 012, India.
ment of cervical mucus penetration and spermegg attachment (Bandivdekar et al., 1987). Previous studies have shown that inhibin has an importance in the physiological function of spermatozoa. The present paper describes the morphometric studies on rat testis after treatment with AshSPI for different durations. The paper also describes the ultrastructural features of the Sertoli cells of normal testis and compares the findings with the fine structure of the Sertoli cells from testes of treated rats.
Materials and methods Preparation of human seminal plasma inhibin (HSPI) The antigen HSPI was purified according to the procedure of Thakur et al. ( 1981) . This homogenous peptide was used to raise the highly specific antibodies by active immunization. The checks for specific antisera were carried out as reported earlier (Shanbaug et al., 1985). These antibodies were used in this study. Adult male rats of Holtzman strain (75 d old; 250-300 g body weight) were subcutaneously injected biweekly with 0.1 ml of either AshSPI or normal rabbit serum (NRS) for 4, 8, and 12 weeks. Animals were killed 2 d after the last injection. The testes were removed and small pieces fixed in modified Karnovsky’s fluid (David et al., 1973), post-fixed in 1% OSO, processed further and embedded in araldite. Semithin sections (0.5 pm) were stained with toluidine blue and observed under bright field optics at a magnification of 400 x and 1000 x . A differential count of cell types was obtained using Chalkley’s point sampling method (1943). Precision was obtained by counting 1000 points over one or more seminiferous tubules. Tubular diameter was measured with an occular micrometer based on 25 tubules/
350
K. GOPALKRISHNAN ET A L .
testis. Care was taken that seminiferous tubules cross sections evaluated, are localized at distant sites in the testis so that they do not represent the same area. Daily sperm production (DSP) For histometric analysis individual seminiferous tubules were separated under a transillumination stereomicroscope at a magnification of 40- 150 x . Tubules measuring 1-2 mm length were dissected, fixed, processed and embedded in araldite. Estimation of DSP was calculated as described earlier (Gopalkrishnan et al., 1987). Hormone estimations Serum FSH and LH were estimated by radioimmunoassay (RIA) using rFSH and rLH kits supplied by NIDDK, M D USA. Electron microscopy Testicular tissue obtained from control (NRS treated) and treated rats were fixed in Karnovsky's fluid for 1 h and postfixed in lo/, OSO, ( 1 h) and after dehydration of samples in acetone the samples were embedded in araldite. Ultrathin sections were cut on a Reichert Ultracut microtome, contrasted with uranyl acetate and lead citrate according to the method of Reynolds (1963). These sections were observed under a Philips 400 EM transmission electron microscope at 80 KV.
Results Morphometry During the present investigation no changes in the testicular weights were observed after different durations of treatment. Daily sperm production as judged by morphometric analysis was reduced b; 50% (Table 1).
Table 1. Tubular diameter and daily sperm production rate in control and in antiserum-treated adult male rats
Table 2. Effect of biweekly administration of 0.1 ml of AshSPI or NRS on circulatory levels of FSH, LH and Prolactin (Prl) in adult male rats (Mean? SE) Hormones
NRS (ng rn1-l)
FSH LH Prl
16k0.3 26k 1.6 204 14.3
AshSPI (ng m1-l) 47+4.1*
25 2.4 205+ 18.4
+
*P
