Seminiferous Tubular Cross Sections Research Articles

O-262 Impact of the cancer treatment received on the quality of the human (pre)pubertal testicular tissue prior to testicular tissue freezing (TTF)

Abstract Study question What is the impact of the cancer treatment received prior to TTF and the disease on spermatogonia quantity in testicular tissue from (pre)pubertal boys? Summary answer A decrease in spermatogonia number was observed in testicular tissue after cancer treatment when cyclophosphamide equivalente dose (CED) is above 4000 mg/m2. What is known already The improved survival rates associated with the development of sperm and testicular tissue freezing (TTF) renders difficult not to offer fertility preservation to children or adolescents before cancer. Several studies exploring cancer patients have examined the number of spermatogonia per seminiferous tubular cross-section (S/T) or tubular fertility index (TFI, percentage of tubular cross-sections containing spermatogonia) in testicular biopsies. All studies, demonstrated that the S/T and TFI always decreased after the introduction of chemotherapy and more specifically in case of highly gonadotoxic risk such as alkylating agents. Study design, size, duration Testicular tissue samples from 79 (pre)pubertal boys diagnosed with cancer (ranging from 6 month to 16 years of age) were cryopreserved between May 2009 and June 2014. Medical diagnosis and previous chemotherapy exposure were recorded. We examined histological sections of (pre)pubertal testicular tissue to elucidate whether chemotherapy, doses or primary diagnosis affects the quality of testicular tissue. Participants/materials, setting, methods (Pre)pubertal boys with cancer diagnosis who benefitted from TTF prior to conditioning treatment for hematopoietic stem cell transplantation. All the patients included had previously received chemotherapy with moderate risk for future fertility. We have selected patients for whom data on chemotherapy received were complete. The quantity of spermatogonia and quality of testicular tissue were assessed by both morphological and immunohistochemical analysis. Main results and the role of chance The main finding was a significant reduction in spermatogonial cell counts in boys treated with alkylating agents. The mean S/T values in boys exposed to alkylating agents was significantly lower than in a group exposed to non-alkylating agents (p = 0.018). In contrast, no difference was observed for patients treated with carboplatin as the only alkylating agent compared to the group of patients exposed to non-alkylating agents. We observed an increase of S/T with age in the group of patients who did not receive alkylating agents and a decrease of S/T with age when patients received alkylating agents included in the CED formula (r = 0.6166, p = 0.0434; r= -0.3759, p = 0.0036, respectively). The TFI and S/T were decreased in the group of patients who received vincristine (p = 0.0049; p < 0.0001, respectively), but the CED was also significantly increased (p < 0.0001). Multivariate analysis, adjusted for CED, showed the persistence of a decrease in TFI correlated with vincristine administration (-0.5 [-0.96; 0.09], p = 0.02). Limitations, reasons for caution This is a descriptive study of testicular tissues obtained from patients who were at risk of infertility. Spermatognia functionanlity could not be tested by transplantation due limited sample size. Wider implications of the findings This study summarizes spermatogonia quantity and quality of testicular tissue of (pre)pubertal boys after potentially sterilizing treatments. We confirmed a negative correlation between the cumulative exposure to alkylating agents and the spermatogonial quantity. For patients in whom fertility preservation is indicated, TTF should be performed before initiation of alkylating agents. Trial registration number N/A

Histological study on the influence of puberty suppression and hormonal treatment on developing germ cells in transgender women.

STUDY QUESTIONCan transgender women cryopreserve germ cells obtained from their orchiectomy specimen for fertility preservation, after having used puberty suppression and/or hormonal treatment?SUMMARY ANSWERIn the vast majority of transgender women, there were still immature germ cells present in the orchiectomy specimen, and in 4.7% of transgender women—who all initiated medical treatment in Tanner stage 4 or higher—mature spermatozoa were found, which would enable cryopreservation of spermatozoa or testicular tissue after having used puberty suppression and/or hormonal treatment.WHAT IS KNOWN ALREADYGender affirming treatment (i.e. puberty suppression, hormonal treatment, and subsequent orchiectomy) impairs reproductive function in transgender women. Although semen cryopreservation is generally offered during the transition process, this option is not feasible for all transgender women (e.g. due to incomplete spermatogenesis when initiating treatment in early puberty, in case of inability to masturbate, or when temporary cessation of hormonal treatment is too disruptive). Harvesting mature spermatozoa, or testicular tissue harboring immature germ cells, from orchiectomy specimens obtained during genital gender-affirming surgery (gGAS) might give this group a chance of having biological children later in life. Previous studies on spermatogenesis in orchiectomy specimens showed conflicting results, ranging from complete absence of germ cells to full spermatogenesis, and did not involve transgender women who initiated medical treatment in early- or late puberty.STUDY DESIGN, SIZE, DURATIONHistological and immunohistochemical analyses were performed on orchiectomy specimens from 214 transgender women who underwent gGAS between 2006 and 2018. Six subgroups were identified, depending on pubertal stage at initiation of medical treatment (Tanner stage 2-3, Tanner stage 4-5, adult), and whether hormonal treatment was continued or temporarily stopped prior to gGAS in each of these groups.PARTICIPANTS/MATERIALS, SETTING, METHODSAll transgender women used a combination of estrogens and testosterone suppressing therapy. Orchiectomy specimen sections were stained with Mayer’s hematoxylin and eosin and histologically analyzed to assess the Johnsen score and the ratio of most advanced germ cell types in at least 50 seminiferous tubular cross-sections. Subsequently, immunohistochemistry was used to validate these findings using spermatogonia, spermatocytes or spermatids markers (MAGE-A3/A4, γH2AX, Acrosin, respectively). Possibilities for fertility preservation were defined as: preservation of spermatozoa, preservation of spermatogonial stem cells or no possibilities (in case no germ cells were found). Outcomes were compared between subgroups and logistic regression analyses were used to assess the association between the duration of hormonal treatment and the possibilities for fertility preservation.MAIN RESULTS AND THE ROLE OF CHANCEMature spermatozoa were encountered in 4.7% of orchiectomy specimens, all from transgender women who had initiated medical treatment in Tanner stage 4 or higher. In 88.3% of the study sample orchiectomy specimens only contained immature germ cells (round spermatids, spermatocytes or spermatogonia, as most advanced germ cell type). In 7.0%, a complete absence of germ cells was observed, all these samples were from transgender women who had initiated medical treatment in adulthood. Cessation of hormonal treatment prior to gGAS did not affect the presence of germ cells or their maturation stage, nor was there an effect of the duration of hormonal treatment prior to gGAS.LIMITATIONS, REASONS FOR CAUTIONSince data on serum hormone levels on the day of gGAS were not available, we were unable to verify if the transgender women who were asked to temporarily stop hormonal treatment 4 weeks prior to surgery actually did so, and if people with full spermatogenesis were compliant to treatment.WIDER IMPLICATIONS OF THE FINDINGSThere may still be options for fertility preservation in orchiectomy specimens obtained during gGAS since a small percentage of transgender women had full spermatogenesis, which could enable cryopreservation of mature spermatozoa via a testicular sperm extraction procedure. Furthermore, the vast majority still had immature germ cells, which could enable cryopreservation of testicular tissue harboring spermatogonial stem cells. If maturation techniques like in vitro spermatogenesis become available in the future, harvesting germ cells from orchiectomy specimens might be a promising option for those who are otherwise unable to have biological children.STUDY FUNDING/COMPETING INTERESTNone.TRIAL REGISTRATION NUMBERN/A.

Gonocyte transformation to spermatogonial stem cells occurs earlier in patients with undervirilisation syndromes

AimFertility post-orchidopexy is dependent on transformation of neonatal gonocytes (G) into adult dark spermatogonia at about 3months, the same time as gonadotrophins stimulate androgen secretion. We examined how androgen blockade affects transformation of gonocytes to spermatogonial stem cells (SSC) during this period in patients with undervirilisation syndromes. MethodsPatients with undervirilisation syndromes (n=30, 1.5weeks–16years) underwent review of medical records, pathology reports, and H&E slides of testes (ethics HREC32164). Fluorescent immunohistochemistry against anti-Mullerian hormone (AMH, Sertoli cells), mouse VASA homologue (MVH, germ cells) and DAPI (nuclei) allowed the number of MVH-positive gonocytes/spermatogonial stem cells per seminiferous tubular cross-section (G/T or SSC/T) to be counted. ResultsGonocytes (MVH-positive cells in the tubular lumen) were present in 15/16 patients under 2 years old. SSC (MVH-positive cells on the tubule basement membrane) were present in 25/30 patients. With increasing age, the mean number of SSC/T decreased from ~4 to 0, and G/T decreased from ~1.5 to 0. SSC were present in CAIS and PAIS patients at 1.5 and 3.5weeks old, respectively. ConclusionsGonocytes transform into SSC earlier than expected in patients with undervirilisation syndromes. Lack of androgens may stimulate non-androgenic regulators to trigger transformation. Understanding how gonocytes transform may enable optimization of spermatogonial development to preserve fertility post-orchidopexy.

Histomorphometric evaluation of the neotropical brown brocket deer Mazama gouazoubira testis, with an emphasis on cell population indexes of spermatogenic yield

Information on the reproductive biology of neotropical cervids is scarce. Therefore, the aim of this study was to perform biometric, histologic and stereologic analyses of the brown brocket deer Mazama gouazoubira testis, with an emphasis on the intrinsic yield and the Sertoli cell index. Seven adult males kept in captivity were used. The animals were immobilized; anesthetized and testicle fragments were obtained by biopsy incision. The material was fixed, processed and examined by routine histological methods for light microscopy. The average body weight was 17.2 kg, from which 0.40% were allocated in gonads and 0.33% in seminiferous tubules, which represented 85.9% of the testis parenchyma. The mean albuginea width and volume were 345.7 μm and 3.5 mL (5.3% of the testicular weight), respectively. The mean mediastinum volume of both testicles was 1.0 mL (1.5% of the testicular weight) and the testicular parenchyma volume corresponded to 93.1% of total testicular weight (64.9 g). The seminiferous tubules diameter was 224.4 μm, while the epithelium height was 69.6 μm. On average, an adult brown brocket deer showed a total of 1418 m of seminiferous tubules in both testicles (21.5 m per gram of testis). Each stage I seminiferous tubular cross section contained 1.10 type A spermatogonia, 13.4 primary spermatocytes in pre-leptotene/leptotene, 13.7 spermatocytes in pachytene, 48.8 round spermatids and 3.7 Sertoli cells. The general yield of spermatogenesis was 44.7 cells and the Sertoli cell index was 13.2. The qualitative and quantitative description of testicular histology of brown brocket deer help to understand its spermatogenic process and to establish parameters for the reproductive biology of this wild species. Furthermore, the data from the present research will help further studies using other species of Brazilian cervids, especially endangered ones, making an additional effort to the species preservation.

Comparative reliability and sensitivity of different methods for assessing treatment effects on sperm production

Investigators may choose among a variety of different approaches for identifying and quantifying potential treatment effects on sperm production rates. Should a treatment actually alter sperm production, the likelihood that the response will be detected and declared statistically significant depends on several factors. These include the size of the treatment response, the inherent variability associated with the endpoint of interest among normal, untreated males, and the number of replicate animals per treatment group. This study was undertaken to characterize the relative power and sensitivity of several widely used methods for quantifying sperm production, for the purpose of identifying those that would be the most likely to enable detection of actual treatment effects. For this exercise, the typical inherent variability associated with several endpoints of testicular function in rats, rabbits and humans was established by a review of the published literature. Based on this typical variability and a components-of-variance approach, the relative number of replicates needed to provide experiments of equivalent power and sensitivity was determined. Consideration was also given to the likely nature of changes in the testis in response to adverse treatments. Based on all of this information, the methods for quantifying spermatogenesis that would be most likely to detect an actual treatment effect were determined to be as follows (from greatest to least powerful and sensitive): (1) quantification of round spermatids per Sertoli cell or per seminiferous tubular cross-section, (2) the determination of the number of spermatids or daily sperm production (DSP) per gram of testis or per testis via volume density approaches, (3) the determination of the number of elongated spermatids or DSP per gram or per testis via the testicular homogenate approach, and (4) the direct enumeration of degenerating germ cells.

Experimental Varicocele Induces Testicular Germ Cell Apoptosis in the Rat

We evaluated the impact of experimentally created varicocele on ipsilateral and contralateral testicular germ cells in the rat. Experimental left varicocele was created by partial ligation of the left renal vein in 17 adult male Sprague-Dawley rats. An additional 5 rats that underwent laparotomy and renal vein handling without ligation served as sham surgical controls. Five rats that underwent no surgical or other intervention served as a control group. Rats were sacrificed 7 (5), 14 (5) or 28 (7) days following varicocele creation. Germ cell apoptosis was quantified using a TUNEL assay. The results of this assay are expressed as the number of apoptotic germ cell nuclei per seminiferous tubular cross section. The presence of apoptosis was confirmed by cellular ultrastucture evaluation using transmission electron microscopy. Control and sham animals were found to have a mean of 0.05 and 0.15 apoptotic germ cells per seminiferous tubular cross section, respectively. Rats sacrificed 7, 14 and 28 days after varicocele creation were found to have 0.15, 0.23 and 0.27 apoptotic germ cells per tubule in the ipsilateral testis, and 0.14, 0.16 and 0.17 apoptotic germ cells per tubule in the contralateral testis, respectively. Compared with control animals a statistically significant increase in the number of apoptotic germ cells per tubular cross section was noted 14 days following varicocele creation in the ipsilateral testis (p <0.05). The creation of experimental varicocele generated an increase in germ cell apoptosis in the ipsilateral testis at 14 days compared with control animals.

Effect of pinealectomy, adrenalectomy, pinealectomy plus adrenalectomy upon the quantification of spermatogenic cells of adult rats

The objectives of this study were to evaluate the effects of pinealectomy, adrenalectomy and pinealectomy-adrenalectomy upon the quantification of spermatogenic cells of rats. As such, 32 adult Wistar rats with a mean body weight of 331.7± 15.5g were assigned into one of the following treatments: (a) a sham-operated control group, consisting of nine animals; (b) ten pinealectomized animals; (c) seven adrenalectomized animals and (d) six pinealectomized plus adrenalectomized animals. No significant differences were observed between groups for the following parameters: body, testes, prostate and seminal vesicle weights, seminiferous tubular diameter, number of cells per seminiferous tubular cross sections (primary spermatocytes at pachytene, round spermatids, Sertoli cells) and numbers of germ cells per Sertoli cell (primary spermatocytes at pachytene and round spermatids ). Although no increase in testicular weight was observed following pinealectomy, a significant (P&lt;0.05) increase of approximately 11.5% in the number of round spermatids per Sertoli cell (Sertoli cell ratio) occurred thus suggesting that short-term pinealectomy abolishes the antigonadal effect of the pineal gland upon adult Wistar rat testes.

Nature of the spermatogenic arrest in Dazl -/- mice.

Dazl encodes an RNA-binding protein essential for spermatogenesis. Mice that are deficient for Dazl are infertile, lacking any formation of spermatozoa, and the only germ cells present are spermatogonia and a few spermatocytes. To gain more insight regarding the timing of the spermatogenic arrest in Dazl -/- mice, we studied the spermatogonial cell types present in testis sections and in seminiferous tubular whole mounts. Most of the seminiferous tubular cross-sections contained A spermatogonia as the most advanced cell type, with only very few containing cells up to pachytene spermatocytes. Both 5-bromodeoxy-uridine incorporation and mitotic index indicated that the remaining A spermatogonia were actively proliferating. C-kit immunohistochemical studies showed that most of the A spermatogonia were positively stained for the c-Kit protein ( approximately 80%). The clonal composition of the A spermatogonia in tubular whole mounts indicated these cells to be A(single) (A(s)), A(paired) (A(pr)), and A(aligned) (A(al)) spermatogonia. It is concluded that the prime spermatogenic defect in the Dazl -/- mice is a failure of the great majority of the A(al) spermatogonia to differentiate into A(1) spermatogonia. As a result, most seminiferous tubules of Dazl -/- mice only contain actively proliferating A(s), A(pr), and A(al) spermatogonia, with cell production being equaled by apoptosis of these cells.

Optimal replication for histometric analyses of testicular function in rats or rabbits

Quantitative evaluations of testicular histology can provide sensitive endpoints for determining toxicity of chemicals to the male reproductive system. But, the numbers of observations per testis or number of animals per treatment group often are selected by tradition or availability, rather than from a statistical basis. Therefore, we studied the number of observations per male (sampling intensity) and number of animals per treatment (replication) needed to detect treatment effects of given magnitude, with predictable error probabilities, using data from Sprague-Dawley rats and Dutch-belted rabbits that received 0.0, 0.94, 1.88, 3.75, 7.5, or 15.0 mg/kg body wt of 1,2-dibromo-3-chloropropane (DBCP). Data for one testis from 102 rats and 34 rabbits were available. For each testis, observations included measurement of the minor diameter of 50 seminiferous tubules, counts of the number of leptotene primary spermatocytes per 250 Sertoli cells, and counts of spherical spermatids within 20 seminiferous tubular cross sections. Tabular data are presented showing optimal numbers of observations per testis and animals per treatment group as a function of the difference to be detected and selected probabilities for Type I and II errors. In general, precise assessments required far fewer observations per testis than are used routinely. However, due to the inherent variability among animals, the number of animals required per treatment tended to be greater for experiments with the rabbit, and increased substantially for both species when detection of small differences had to be ensured. The data presented should enable investigators to design experiments of chosen sensitivity and precision while making cost-effective use of animals and labor.

Sampling Intensities and Replication Requirements for Detection of Treatment Effects on Testicular Function in Bulls and Stallions: A Statistical Assessment

Data from testes of 16, 2- to 3-yr-old stallions and 34 yearling beef bulls were utilized in a components of variance approach to calculate the number of observations required per testis and(or) the number of animals required per treatment group to provide experiments of known sensitivity and precision, where treatment was to be assessed by one of several endpoints. The latter included paired testes weight, seminiferous tubular diameter, the number of germ cells per seminiferous tubular cross-section, or the number of elongated spermatids per gram of testicular parenchyma or per testis. For all variables for which several observations were available for each testis, precise assessment of any given male required far fewer observations than have been used routinely. However, replication requirements varied substantially in relation to the sensitivity (i.e., size of difference to be detected) and precision (i.e., Type I and II error probabilities) desired. Replication requirements were greater for stallions than for bulls, particularly at higher levels of sensitivity, for which requirements for both species were very large. The data presented should permit future experiments involving assessment of these endpoints to be designed with known sensitivity and precision and with optimal efficiency and cost-effectiveness.

The numbers of Sertoli cells in mature Holstein bulls and their relationship to quantitative aspects of spermatogenesis.

Testes from 37 Holstein bulls, 38-99 mo of age, were used to investigate the relationship of Sertoli cell number, Sertoli cell-germ cell ratios and other related factors to daily sperm production (DSP). DSP was assessed by enumeration of spermatids in testicular homogenates, whereas Sertoli cell and germ cell ratios were based on direct counts in 20 round Stage VIII seminiferous tubular cross sections per bull. Numbers of Sertoli cells were calculated as (total homogenization resistant spermatids:spermatid:Sertoli cell ratio)/0.394; the factor of 0.394 adjusted for the presence of homogenization resistant spermatids during only 39.4% of the spermatogenic cycle. Data were subjected to simple linear and second-order regression analyses. Positive linear relationships were observed between DSP and testicular parenchymal weight (p less than 0.005, R = +0.71), DSP per gram (p less than 0.005, R = +0.79), total Sertoli cells (p less than 0.005, R = +0.83), Sertoli cells per gram (p less than 0.01, R = +0.47) and the yield of Step 8 spermatids per Type A spermatogonium (p less than 0.05, R = +0.34). DSP was not related (p greater than 0.10) to the number of germ cells supported per Sertoli cell. Testicular parenchymal weight and DSP per gram were unrelated to each other (p greater than 0.10), but both were related (p less than 0.005) to the total Sertoli cell number (R = +0.61 and +0.62, respectively). Total number of Sertoli cells accounted for more of the variation in DSP between bulls (R2 = 68.2%) than did any other factor examined. It was suggested that total Sertoli cell number may be an important determinant of a bull's spermatogenic potential.

Relationship of absolute numbers of Sertoli cells to testicular size and spermatogenesis in young beef bulls.

Testes were obtained from 34 Hereford or Angus bulls at about 1.5 yr of age and were used to investigate the relationship between the absolute number of Sertoli cells vs testicular size and daily spermatozoal production (DSP). Quantitative determination of DSP was based upon enumeration of elongated spermatids in testicular homogenates. The ratio of step 8 spermatids to Sertoli cells (S:SC) was established by direct counts of these cells in each of 20 round stage VIII seminiferous tubular cross sections for each bull. The number of Sertoli cells per paired testes was calculated as (total spermatids divided by S:SC)/.394, where total spermatids equalled the number of homogenization-resistant spermatids. The factor of .394 adjusted for the fact that the latter cells are present for only 39.4% of the spermatogenic cycle. All data were subjected to simple linear and second-order regression analyses. A positive linear relationship (P less than .005) was found between testicular weight (Y, in grams) and the absolute number of Sertoli cells per paired testes (X, in billions), which was characterized by the equation Y = 315.2 + 10.74X and a coefficient of correlation (r) of .56 (P less than .01). A similar relationship was observed between DSP (Y, in billions) and Sertoli cell numbers (X, in billions). This was characterized by the equation Y = 1.36 + .222X (P less than .005) and a coefficient of correlation of .70 (P less than .01). Daily sperm production was unrelated to the S:SC ratio (P greater than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Use of quantitative testicular histology to assess the effect of dibromocholoropropane (DBCP) on reproduction in rabbits

Dibromochloropropane (DBCP) was administered orally to 36 sexually mature male Dutch Belted rabbits assigned at random to one of six groups to receive 0, 0.94, 1.88, 3.75, 7.5, or 15.0 mg DBCP per kilogram of body weight daily 5 days per week during a 69-day treatment period. Animals were euthanized and necropsied on Day 70. Body weights and weights of the kidneys, liver, epididymides, and accessory sex glands were not influenced by DBCP treatment. The highest dosage reduced mean paired testes weight to 45% of control values ( p < 0.01). Mean seminiferous tubular diameter was reduced by 15 and 29% with the two highest dosages of DBCP, respectively ( p < 0.01), and the percentage of seminiferous tubules containing elongating and round spermatids, as the most advanced cell type present, was decreased ( p < 0.01). The number of leptotene primary spermatocytes per Sertoli cell, and the numbers of spermatogonia, young and old primary spermatocytes, and Step 1 spermatids per Stage I seminiferous tubular cross section likewise indicated a general depression ( p < 0.01) of spermatogenesis. Based upon the number of Step 1 spermatids per cross section at Day 70, production of sperm at this time by rabbits receiving the highest dose of DBCP was estimated to be less than one-fourth of control values. No significant effect ( p > 0.05) of 0.94 mg DCBP/kg on any parameters was found, so this could be considered to be the no effect dose. However, the regressions and mean values are interpreted to indicate that the no effect level of DBCP may be less than 0.94 mg DBCP/kg of body weight. It is suggested that the most sensitive tests described here be included in future screening tests for potential effects of agents on male reproduction.

Dietary influences on theobromine-induced toxicity in rats

Theobromine-induced toxicity was compared at 8, 16, 21, and/or 28 days in rats given either a semisynthetic diet or a pulverized commercial diet in each of which 0.6 or 0.8% theobromine was incorporated. Rats given the pulverized commercial diet containing theobromine consumed more food and therefore, more theobromine over a 28-day period, and gained more weight than did rats given theobromine incorporated into the semisynthetic diet. Serum theobromine concentrations were significantly higher in rats given 0.8% theobromine in the semisynthetic diet than in rats given 0.8% theobromine in the pulverized commercial diet. Spermatogenic cell degeneration and necrosis in the testes of rats fed 0.8% theobromine in the semisynthetic diet for 16 days seemed to be limited to seminiferous tubular cross sections containing stages X to XIV of spermatogenesis, but after 28 days all tubular cross sections showed extensive spermatogenic cell destruction. No significant pathological changes were observed in the testes of rats fed 0.8% theobromine in the pulverized commercial diet for 21 days; spermatogenic cell degeneration and necrosis and multinucleate cell formation were seen after 28 days in seminiferous tubular cross sections containing the early stages of spermatogenesis, but sections containing stages X to XIV were spared or were much less involved. Atrophy of the thymus gland occurred earlier than testicular damage on each respective dietary regimen containing theobromine and was more severe in rats given theobromine in the semisynthetic diet.

Unregelmässig aufgebaute Zellgemeinschaften des Samenepithels beim free-ranging japanischen Makak (Macaca fuscata) in der Paarungszeit

Testicular biopsies were obtained from 11 adult males of free-ranging Japanese macaques trapped from Takasakiyama troops at the height of the breeding season. The spermatogenesis was observed on Bouin fixed and hematoxylin-eosin stained sections, especially from the viewpoint of seminiferous epithelial cycle. One cycle may be classified into seven stages. The classification, however, is not very easily carried out, because the seminiferous epithelium is, in most cases, very irregularly constructed, and typical germ cell associations which are easily identified, are infrequently observed. On the basis of the classification, various types of irregularities in the arrangement of the cellular associations were investigated and classified. In an unexpectedly large number of seminiferous tubular cross-sections, the lumina are obscured or obliterated owing to disarrangement of germ cells. As a consequence of this, the tubular sections with clearly discernible lumina are very rarely found (38 in a total of 1,215 observed cross-sections). Furthermore, the presence of several different stages in a singular cross-section is rather a common phenomenon (404 in a total of 1,215).