NR5A1 is required for functional maturation of Sertoli cells during postnatal development

The orphan nuclear receptor steroidogenic factor 1 (NR5A1 (SF-1)) is expressed in both Sertoli and Leydig cells in the testes. This study investigates the postnatal development of the testes of a gonad-specific Nr5a1 knockout (KO) mouse, in which Nr5a1 was specifically inactivated. The KO testes appeared histologically normal from postnatal day 0 (P0) until P7. However, disorganized germ cells, vacuoles, and giant cells appeared by P14 in the seminiferous tubules of KO but not control mice. Expression of NR5A1 and various factors was examined by immunohistochemistry (IHC). The number of NR5A1-positive Sertoli cells in the KO testes was lower compared with controls at all the developmental stages and decreased to nearly undetectable levels by P21. IHC for anti-Müllerian hormone and p27, immature and mature Sertoli cell markers, respectively, indicated a delay in Sertoli cell maturation in the KO testes. The number of Sertoli cell-expressing factors involved in Sertoli cell differentiation including WT1, SOX9, GATA4, and androgen receptor were lower in the KO testes compared with controls. Furthermore, fewer proliferating cell nuclear antigen-positive proliferative germ cells were observed, and the number of TUNEL-labeled cells was significantly higher in the KO testes compared with controls at P14 and P21, indicating impaired spermatogenesis. IHC for CYP11A1 (SCC) indicated the presence of steroidogenic Leydig cells in the interstitium of the KO testes at all stages examined. These results suggest that NR5A1 is essential for Sertoli cell maturation and therefore spermatogenesis, during postnatal testis development.     

Changes of testicular aromatase expression during fetal development in male pigs (Sus scrofa)

Male pig fetuses secrete considerable amounts of estrogens, but the location of aromatase activity within the fetal testis is not known. The location of aromatase expression was investigated by immunocytochemistry in fetal testes from week 6 (n = 5), weeks 10, 13, and 15 (each: n = 6) of gestation and additionally in neonates (n = 4). Blood was sampled from the umbilical artery of fetuses and jugular vein of neonates. Histological evaluation of testes involved morphological criteria and counting of Leydig cells, Sertoli cells, and gonocytes. Aromatase activity was localized immunocytochemically and quantified by the percentage of positive stained cells within the same cell type. Aromatase expression was further characterized by quantitative RT-PCR. Concentrations of estrogens, testosterone, FSH, and LH were measured in blood plasma. Total estrogens increased from week 10 to a maximum of 31.03 nmol/l in week 15. Increased testosterone concentrations were only measured at week 6 and were paralleled by slightly elevated estrogens. Thereafter, testosterone dropped and was low throughout. The increase of estrogens was not paralleled by a similar increase of FSH and LH but was related to the increase of the total number of Leydig cells. This increase was also found for mRNA expression. Both Leydig cells and gonocytes were identified as contributors to estrogen formation. Gonocytes were the main source of aromatase at week 10, when gene expression by Leydig cells is low due to the preparation of a wave of Leydig cell mitosis.     

Inhibin, activin, follistatin and FSH serum levels and testicular production are highly modulated during the first spermatogenic wave in mice

Testicular development is governed by the combined influence of hormones and proteins, including FSH, inhibins, activins and follistatin (FST). This study documents the expression of these proteins and their corresponding mRNAs, in testes and serum from mice aged 0 through 91 days post partum (dpp), using real-time PCR, in situ hybridisation, immunohistochemistry, ELISA and RIA. Serum immunoactive total inhibin and FSH levels were negatively correlated during development, with FSH levels rising and inhibin levels falling. Activin A production changed significantly during development, with subunit mRNA and protein levels declining rapidly after 4 dpp, while simultaneously levels of the activin antagonists, FST and inhibin/activin beta(C), increased. Inhibin/activin beta(A) and beta(B) subunit mRNAs were detected in Sertoli, germ and Leydig cells throughout testis development, with the beta(A) subunit also detected in peritubular myoid cells. The alpha, beta(A), beta(B) and beta(C) subunit proteins were detected in Sertoli and Leydig cells of developing and adult mouse testes. While beta(A) and beta(B) subunit proteins were observed in spermatogonia and spermatocytes in immature testes, beta(C) was localised to leptotene and zygotene spermatocytes in immature and adult testes. Nuclear beta(A) subunit protein was observed in primary spermatocytes and nuclear beta(C) subunit in gonocytes and round spermatids. The changing spatial and temporal distributions of inhibins and activins indicate that their modulated synthesis and action are important during onset of murine spermatogenesis. This study provides a foundation for evaluation of these proteins in mice with disturbed testicular development, enabling their role in normal and perturbed spermatogenesis to be more fully understood.     

Expression and localization of inhibin alpha,inhibin/activin betaA and betaB and the activin type II and inhibin beta-glycan receptors in the developing human testis

Inhibins and activins have roles in the regulation of cell proliferation and differentiation in a variety of tissues. This study investigated the distribution of the three inhibin/activin subunits (alpha, betaA and betaB) and their receptors in the human testis between week 13 and week 19 of gestation using RT-PCR and immunohistochemistry. mRNA for all three subunits and for the activin type II receptors ActRIIA and ActRIIB was detected at all stages of gestation examined. Sertoli cells showed intense immunostaining for the alpha subunit and some staining for the betaB subunit, whereas only the betaB subunit was detected in gonocytes. No betaA subunit staining was detected within the tubules. All three subunits were localized to interstitial Leydig cells. Cells of the rete testis and the epididymal epithelium also showed immunostaining for betaB; however, staining for the other subunits was weak or absent. Peritubular cells showed intense immunostaining for the beta-glycan inhibin receptor, which was also localized to interstitial cells, but was not detected within the tubular compartment, rete testis or epididymal epithelium. ActRIIA was detected in gonocytes and in interstitial cells; ActRIIB was distributed widely. These data indicate that fetal Leydig and Sertoli cells have the potential to produce both activins and inhibins, whereas gonocytes may produce only activin B. The distribution of activin and inhibin receptors implies that the intratubular compartment and developing duct system are sites of action of activin B but not inhibin at this stage of development, whereas both activins and inhibins may be involved in the development and function of the peritubular and interstitial cells.     

The effects of oestrogen receptors alpha and beta on testicular cell number and steroidogenesis in mice

Oestrogen plays an important role in testicular function. This study used mice null for oestrogen receptor alpha (ER alpha) or beta (ER beta) to investigate which receptor mediates the effects of oestrogen within the testis. Groups of ER alpha knockout mice (alpha ERKO) and ER beta knockout mice (beta ERKO) and wild-type littermates (n=5-8) were killed at 11 weeks post partum. One testis was fixed in Bouin's fluid for stereology and the other frozen for testosterone measurement. Trunk blood was collected for testosterone RIA. The optical disector combined with the fractionator methodology was used to estimate Leydig, Sertoli and germ cell numbers. At all times, the knockout animals were compared with their wild-type littermates. The physical disector quantified cells stained immunohistochemically for the apoptotic marker active caspase-3 and Hoechst staining was used to identify nuclear fragmentation. The mean Leydig cell volume was measured using the point sampled intercept method. The Leydig cell number per testis was significantly increased in beta ERKO mice but not in alpha ERKO mice. Plasma and testicular testosterone concentrations were increased in alpha ERKO mice but no changes were observed in beta ERKO mice. Hypertrophic Leydig cell changes were observed in alpha ERKO mice, and a decreased mean cell volume was seen in beta ERKO mice. No difference in Sertoli cell number per testis was observed in any of the groups. The spermatogonial cell number per testis was increased in beta ERKO mice. Immunohistochemistry identified increased numbers of active caspase-3-labelled germ cells per testis in alpha ERKO mice but not beta ERKO mice. Hoechst staining supported these findings. There was significant germ cell loss in alpha ERKO mice. This study suggests that ER beta may be involved in regulation of Leydig cell proliferation and testosterone production in the adult mouse testis.     

Use of antibodies against LH receptor, 3beta-hydroxysteroid dehydrogenase and vimentin to characterize different types of testicular tumour in dogs

Testicular tumours in dogs are of Sertoli cell, Leydig cell or germinal origin and mixed tumours are also frequently observed. The cellular components of mixed tumours are usually identified by histological examination but sometimes this is difficult. In this study, a panel of specific antibodies was used to identify the different cell types in testicular tumours by immunohistochemistry. Leydig cells were identified using an antibody against the LH receptor and an antibody against the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD), both of which are characteristic of Leydig cells in testes. Sertoli cells were identified using an antibody against the intermediate filament vimentin. Seminoma cells did not stain with any of these antibodies. Vimentin was used only in histologically complex cases. Eighty-six tumours, diagnosed histologically as 29 Sertoli cell tumours, 25 Leydig cell tumours, 19 seminomas and 13 mixed tumours, were studied. Feminization was observed in 17 dogs. Leydig cell tumours stained positively with the antibodies against the LH receptor and 3beta-HSD, whereas seminomas and Sertoli cell tumours were negative (unstained). The antibody against vimentin stained both Sertoli and Leydig cells, and tumours arising from these cells, but not seminomas. Immunohistochemistry revealed that three tumours identified histologically as Sertoli cell tumours were actually Leydig cell tumours. In 14 dogs the histological diagnosis appeared to be incomplete, as mixed tumours instead of pure types of tumours were identified in 11 dogs, and in three dogs mixed tumours appeared to be pure types. Hence, the histological diagnosis was insufficient in approximately 20% of dogs. Furthermore, immunohistochemical analysis of testis tumours revealed that feminization occurred in dogs with Sertoli cell tumours or Leydig cell tumours and their combinations, but not in dogs with a seminoma. In conclusion, incubation with antibodies against LH receptor and 3beta-HSD proved to be a consistently reliable method for identification of Leydig cell tumours in dogs. Vimentin can be used to discriminate between Sertoli cell tumours and seminomas. Overall, this panel of antibodies can be very useful for determination of the identity of testicular tumours in which histological characterization is complicated and the pathogenesis of feminization is not clear.     

Expression of steroidogenic enzymes during equine testicular development

In the mammalian testis, Leydig cells are primarily responsible for steroidogenesis. In adult stallions, the major endocrine products of Leydig cells include testosterone and estrogens. 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4)-isomerase (3βHSD) and 17α-hydroxylase/17,20-lyase (P450c17) are two key steroidogenic enzymes that regulate testosterone synthesis. Androgens produced by P450c17 serve as substrate for estrogen synthesis. The aim of this study was to investigate localization of the steroidogenic enzymes P450c17, 3βHSD, and P450arom and to determine changes in expression during development in the prepubertal, postpubertal, and adult equine testis based upon immunohistochemistry (IHC) and real-time quantitative PCR. Based on IHC, 3βHSD immunolabeling was observed within seminiferous tubules of prepubertal testes and decreased after puberty. On the other hand, immunolabeling of 3βHSD was very weak or absent in immature Leydig cells of prepubertal testes and increased after puberty. HSD3B1 (3βHSD gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal testes (P=0.0041). P450c17 immunolabeling was observed in small clusters of immature Leydig cells in prepubertal testes and increased after puberty. CYP17 (P450c17 gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.030) and postpubertal testes (P=0.0318). A weak P450arom immunolabel was observed in immature Leydig cells of prepubertal testes and increased after puberty. Similarly, CYP19 (P450arom gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal (P=0.0001) testes. In conclusion, Leydig cells are the primary cell type responsible for androgen and estrogen production in the equine testis.     

Stimulation of Sertoli cell proliferation: defining the response interval to an inhibitor of estrogen synthesis in the boar

Sertoli cell proliferation occurs in two major waves after birth, one neonatally and another prepubertally, each contributing to final testicular size and sperm production. However, little is known about the regulation of either wave. We have previously shown that letrozole, an inhibitor of estrogen synthesis, increases Sertoli cell number and testicular size at sexual maturity in boars. These studies were conducted to determine whether letrozole affects the first or second proliferative wave. Boars were treated with letrozole during the first wave (treatment at 1, 3, and 5 weeks), less frequently (1 week of age only, or 1 and 5 weeks), on postnatal day 1, or during the second wave (weeks 11-16). Sertoli cells were enumerated in testes and estrogen concentrations were evaluated in serum and testes. Compared with vehicle controls, letrozole reduced estrogen in boars treated at weeks 1 and 5 or 1, 3, and 5, on postnatal day 1, or prepubertally. However, Sertoli cell numbers were increased only in boars treated at 1, 3, and 5 weeks of age. Neither perinatal (1 day old) nor prepubertal letrozole treatment affected Sertoli cell numbers. Hence, Sertoli cell proliferation was sensitive to letrozole only if letrozole was administered throughout the first wave, even though estrogen synthesis was effectively inhibited at all ages. These data indicate that the neonatal but not the prepubertal window of Sertoli cell proliferation is sensitive to an inhibitor of estrogen synthesis; this suggests that these two waves are differently regulated.     

Spermatogonial morphology and kinetics during testis development in mice: a high-resolution light microscopy approach

Despite the knowledge of spermatogonial biology in adult mice, spermatogonial development in immature animals has not been fully characterized. Thus, the aim of this study was to evaluate the ontogeny of the morphological development of the spermatogonial lineage in C57BL/6 mouse testis, using high-resolution light microscopy. Spermatogonial morphology, chronology, and absolute number were determined for different ages postpartum (pp). The morphology of spermatogonia in immature mice was similar to that of adult spermatogonia, although their nuclear diameter was slightly smaller. The A(1) spermatogonia were first observed on day 2 pp, and only 24 h later, differentiating type A(3) and A(4) spermatogonia were observed in the seminiferous cords. This result indicated a shortening of the spermatogonial phase for immature mice of about ～2.5 days when compared with adult mice and suggests that gonocytes and/or A(1) spermatogonia could directly become A(4) spermatogonia, skipping the developmental sequence of type A spermatogonia. These A(4) spermatogonia are functional as they develop into type B spermatogonia by day 5 pp. At day 8 pp, while differentiation to spermatocytes begins, the A(und) spermatogonia reach their maximal numbers, which are maintained through adulthood. The various details of the spermatogonial behavior in immature normal mice described in this study can be used as a baseline for further studies under experimental or pathological conditions.     

Endocrine differentiation of fetal ovaries and testes of the spotted hyena (Crocuta crocuta): timing of androgen-independent versus androgen-driven genital development

Female spotted hyenas (Crocuta crocuta) have an erectile peniform clitoris and a pseudoscrotum but no external vagina, all established by day 35 of a 110-day gestation. Recent studies indicate that these events are androgen-independent, although androgen secretion by fetal ovaries and testis was hypothesized previously to induce phallic development in both sexes. We present the first data relating to the capacity of the ovaries and testes of the spotted hyena to synthesize androgens at different stages of fetal life. Specifically, spotted hyena fetal gonads were examined by immunohistochemistry at GD 30, 45, 48, 65, and 95 for androgen-synthesizing enzymes, as related to the morphological development. Enzymes included 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), cytochrome b5, 3beta-hydroxysteroid dehydrogenase (3betaHSD), and cholesterol side-chain cleavage cytochrome P450 (P450scc). Anti-Müllerian-hormone (AMH) expression was also examined. AMH was strongly expressed in fetal Sertoli cells from GD 30 and after. P450c17 expression was detected in Leydig cells of developing testes and surprisingly in Müllerian duct epithelium. Fetal ovaries began to organize and differentiate by GD 45, and medullary cells expressed P450c17, cytochrome b5, 3betaHSD, and P450scc. The findings support the hypothesis that external genital morphology is probably androgen-independent initially, but that fetal testicular androgens modify the secondary, male-specific phallic form and accessory organs. Fetal ovaries appear to develop substantial androgen-synthesizing capacity but not until phallic differentiation is complete, i.e. after GD 45 based on circulating androstenedione concentrations. During late gestation, fetal ovaries and testes synthesize androgens, possibly organizing the neural substrates of aggressive behaviors observed at birth in spotted hyenas. These data provide an endocrine rationale for sexual dimorphisms in phallic structure and reveal a potential source of androgenic support for neonatal aggression in female and male C. crocuta.     

Dose-response of RAG2-/-/gammac-/- mice to busulfan in preparation for spermatogonial transplantation

Practical applications of spermatogonial transplantation require good rates of colonization by the donor cells. Recipient testes are usually depleted of competing endogenous spermatogonia by administration of 32-44 mg busulfan kg(-1) body weight before transplantation. However, it is not clear that this is the optimum dose, especially for immunodeficient mice. In the present study, the response of adult RAG2(-/-)/gamma(c)(-/-) (RAG2) male mice to treatment with 10-50 mg busulfan kg(-1) body weight was determined in terms of mortality rates, testicular masses and histology, and colonization of seminiferous tubules by transplanted spermatogonia. Mortality increased from 0 to 50% at doses between 20 mg busulfan kg(-1) and 40 mg busulfan kg(-1), whereas the maximum effects on testicular mass and histology were observed at 20 mg busulfan kg(-1). Colonization of testes by genetically marked spermatogonia after treatment of mice with 20 mg busulfan kg(-1) was equivalent to rates previously reported in recipients treated with 32-44 mg busulfan kg(-1). Thus, 20 mg busulfan kg(-1) appears to be the optimum dose for preparing RAG2 mice for spermatogonial transplantation. However, because the steepness of the dose-response curves indicates that direct administration of busulfan is not ideal for this purpose, 15 mg busulfan kg(-1) was administered to pregnant females at various times between day 10.5 and day 16.5 of gestation to determine whether this would deplete the number of germ cells in male offspring. Although there were large variations in testicular mass and histology, no mortality was observed and administration of busulfan at day 10.5 or 12.5 after mating delayed initiation of spermatogenesis, indicating that prenatal administration of busulfan combined with neonatal transplantation might be an effective method for further increasing rates of colonization by donor spermatogonia.     

A close correlation in the expression patterns of Af-6 and Usp9x in Sertoli and granulosa cells of mouse testis and ovary

Usp9x, an X-linked deubiquitylating enzyme, is stage dependently expressed in the supporting cells (i.e. Sertoli cells and granulosa cells) and germ cells during mouse gametogenesis. Af-6, a cell junction protein, has been identified as a substrate of Usp9x, suggesting a possible association between Usp9x and Af-6 in spermatogenesis and oogenesis. In this study, we examined the expression pattern of Af-6 and Usp9x and their intracellular localization in testes and ovaries of mice treated with or without pregnant mare serum gonadotropin (PMSG), an FSH-like hormone. In both testes and ovaries, Af-6 expression was predominantly observed in supporting cells, as well as in steroidogenic cells, but not in any germ cells. In Sertoli cells, Af-6 was continuously expressed throughout postnatal and adult stages, where both Af-6 and Usp9x were enriched at the sites of Sertoli-Sertoli and Sertoli-spermatid junctions especially at stages XI-VI. In the granulosa cells, Af-6, as well as Usp9x, was highly expressed in primordial and primary follicles, but its expression rapidly decreased after the late-secondary follicle stage. Interestingly, in PMSG-treated mice, the expression levels of Af-6 and Usp9x were synchronously enhanced, slightly in Sertoli cells and strongly in granulosa cells of the late-secondary and Graafian follicles. Such closely correlated expression patterns between Af-6 and Usp9x clearly suggest that Af-6 may be deubiquitylated by Usp9x in both Sertoli and granulosa cells. It further suggests that the post-translational regulation of Af-6 by Usp9x may be one potential pathway to control the cell adhesion dynamics in mammalian gametogenesis.     

Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis

Germ cell proliferation, migration and survival during all stages of spermatogenesis are affected by stem cell factor signalling through the c-Kit receptor, the expression and function of which are vital for normal male reproductive function. The present study comprehensively describes the c-Kit mRNA and protein cellular expression profiles in germ cells of the postnatal and adult rodent testis, revealing their significant elevation in synthesis at the onset of spermatogenesis. Real-time PCR analysis for both mice and rats matched the cellular mRNA expression profile where examined. Localization studies in normal mouse testes indicated that both c-Kit mRNA and protein are first detectable in differentiating spermatogonia. In addition, all spermatogonia isolated from 8-day-old mice displayed detectable c-Kit mRNA, but 30-50% of these lacked protein expression. The c-Kit mRNA and protein profile in normal rat testes indicated expression in gonocytes, in addition to differentiating spermatogonia. However, in the irradiated adult rat testes, in which undifferentiated spermatogonia are the only germ cell type, mRNA was also detected in the absence of protein. This persisted at 3 days and 1 and 2 weeks following treatment with gonadotrophin-releasing hormone (GnRH) antagonist to stimulate spermatogenesis recovery. By 4 weeks of GnRH antagonist treatment, accompanying the emergence of differentiating spermatogonia, both mRNA and protein were detected. Based on these observations, we propose that c-Kit mRNA and protein synthesis are regulated separately, possibly by influences linked to testis maturation and circulating hormone levels.     

Leydig cell function in mice lacking connexin43

Connexin43 (Cx43) is the most abundantly expressed member of the connexin (gap junction protein) family and the only one so far identified in mouse Leydig cell gap junctions. Mice lacking Cx43 were used to investigate its role in testicular androgen production and regulation. Testes from term fetuses were grafted under the kidney capsules of castrated adult males. After 3 weeks, serum from host mice was analyzed for androgens. In order to test their response to stimulation, the grafted testes were incubated in vitro with varying concentrations of LH and their androgen end products analyzed. Incubation with radiolabeled progesterone was followed by high performance liquid chromatography to quantify the androgen-intermediate metabolites. Radiolabeled testosterone in the presence of NADPH was used to determine the activity of testosterone-metabolizing enzymes 17beta-hydroxysteroid dehydrogenase (17betaHSD), 5alpha-reductase (5alphaR), and 3alpha-hydroxysteroid dehydrogenase (3alpha HSD). Serum androgen levels did not differ between hosts carrying wild-type versus null mutant grafts although Cx43-deficient testes had more 17betaHSD and 5alphaR activity than wild-type controls. Furthermore, the genotype of grafted testes did not influence LH-stimulated androgen production in vitro. These results indicate that the steroidogenic function of Leydig cells is not compromised by the absence of Cx43, perhaps because other gap junction proteins are present. Dye transfer experiments demonstrated that Cx43-deficient Leydig cells retain intercellular coupling, indicating that Cx43 is not the only protein contributing to their gap junctions. Thus, despite their prominence in Leydig cells, Cx43 gap junctions are not essential for androgen production.     

Endocrine changes associated with onset of spermatogenesis in Holstein bulls

After birth, a bull enters a period of infancy during which the reproductive organs are relatively quiescent. This is followed by the prepubertal period, which starts at 10 to 12 wk in well-fed Holstein bulls, characterized by profound changes of hypothalamic, pituitary, and gonadal function that culminate in puberty. The prepubertal sequence of events probably is: a) initiation of spontaneous discharge of luteinizing hormone; b) hormone induced differentiation of Leydig cells with increased secretion of androstenedione in response to luteinizing hormone stimulation; c) further differentiation of Leydig cells resulting in luteinizing hormone-stimulated secretion of testosterone; d) testosterone-induced differentiation of indifferent supporting cells to Sertoli cells concomitant with testosterone-induced differentiation of gonocytes to prespermatogonia and A-spermatogonia; e) increased sensitivity of the hypothalamus-anterior pituitary to negative feedback of gonadal steroids; f) diminished frequency and amplitude of luteinizing hormone discharge; g) formation of junctional complexes between Sertoli cells and establishment of the blood-testis barrier; h) formation of primary spermatocytes and ultimately spermatids and spermatozoa; and i) continued increase of efficiency of spermatogenesis until sufficient sperm are produced to provide the first ejaculum around 37 to 38 wk. Following puberty, the reproductive capacity of a bull increases for several years until the male is sexually mature.     

Arrested apoptosis without nuclear fragmentation produced by efferent duct ligation in round spermatids and multinucleated giant cells of rat testis

The apoptotic process evoked by efferent duct ligation in the testes of adult rats was followed for 10 days by differential staining for haematoxylin-eosin, periodic acid-Schiff and a modified trichrome technique in optical microscopy and by ultrastructural localization of acid phosphatase. Round spermatids showed the first effects of efferent duct ligation. At day 3 after ligation, annular clumps of chromatin with typical apoptotic characteristics appeared against the nuclear membrane of these cells. Afterwards, membranous structures and a wide separation between the two layers of the nuclear membrane were observed but nuclear fragmentation did not occur and apoptotic granules were not seen. Cytoplasmic components were also altered, and severely damaged organoids and empty vacuoles lacking acid phosphatase reaction were frequently seen. On day 2 after efferent duct ligation, multinucleated giant cells appeared, which displayed similar characteristics as spermatids and showed no acid phosphatase reaction. Although abnormal spermatids and multinucleated giant cells were surrounded by the cytoplasm of Sertoli cells, neither lysosomal acid phosphatase nor phagocytic activity was detected. It is concluded that efferent duct ligation specifically affects round immature spermatids eliciting a partial nuclear apoptotic response that is not accompanied by autophagic or heterophagic activity and without lysosomal participation in Sertoli cells.