Abstract
The cynomolgus monkey (
Introduction
The strong phylogenetic relationship of monkeys (both New and Old World) to man makes nonhuman primates very suitable as models to study the effects on the reproductive system. Therefore, cynomolgus, rhesus, and marmoset monkeys are regularly used in drug development programs, especially when new hormonal drugs are tested. For such compounds the dog is less suitable as the nonrodent species. Male monkeys are particularly used in studies involving compounds that are developed in the field of male contraception and hormone therapy. In these studies, the male reproductive tract is a potential target system and one of the important systems to be evaluated for pharmacological activity and toxicity. Histopathology is acknowledged as a sensitive endpoint for detecting testicular toxicity (Creasy, 1997). For this purpose it is essential to distinguish drug-induced histological changes from the normal variation. Therefore, an adequate understanding of the organization and dynamics of the process of spermatogenesis of the test animal used is a prerequisite (Foley, 2001; Lanning et al., 2002). Unfortunately, detailed and up-to-date information on the general organization of the testes in monkeys is limited.
Spermatogenesis, the fundamental function of the testis, is a complex continuum of cellular differentiation. In the rhesus monkey, spermatogenesis has already been described extensively by Clermont and Leblond (1959). Even today, this publication is still used by researchers as a guide for detailed histopathological evaluation of the monkey testis in general. This publication describes 12 different stages of the seminiferous epithelium in cross-sectioned testis tubules. Each stage is defined as the grouping (association) of various germ cell types at a specific time point of development. Morphological criteria for the different stages are mainly based upon the development of the spermatids.
It is generally assumed that the process of spermatogenesis in the cynomolgus monkey is highly comparable to that in men. Clermont (1963) described 6 stages in the process of spermatogenesis in human. Cross-sections of individual seminiferous tubules in human testis may contain one or more spermatogenic stages with intermingling of germ cells at the borders of adjacent stages. In marmosets such multistage cross-sections are observed as well (Millar et al., 2000; Li et al., 2005).
The goal of the present paper is to describe spermatogenesis and the cellular composition of the 12 stages of the spermatogenic cycle (utilizing the classification scheme of Clermont and Leblond, 1959) in the sexually mature cynomolgus monkey testes and to provide clear illustrative photomicrographs of the individual stages in sections of routinely processed tissues. In addition, the features of sexually immature and prepubertal monkey testes are discussed with respect to the potential confusion of these features with treatment-related changes in toxicity studies.
This practical approach for testis staging in cynomolgus monkeys may be helpful for toxicological pathologists in identifying the various stages and detecting abnormalities. Microscopic evaluation should be carried out as a qualitative examination with the awareness of the spermatogenic cycle. Quantitative measurements of tubular stages and their frequency distribution are not recommended for screening studies (Lanning et al., 2002).
Materials and Methods
In the present paper, vehicle-treated cynomolgus monkeys have been used from different toxicity studies and sources. The mature cynomolgus monkeys (Mauritian origin) were purpose-bred and obtained from an internationally recognized supplier. In total 4 animals were chosen from the placebo group of a toxicity study. The monkeys were 5–8 years old and their body weights ranged from 4.0 to 7.0 kg. Sexual maturity was assessed during the pre-dose period by evaluating sperm in semen samples, testes volume, LH- and testosterone levels. Testosterone levels were determined from plasma samples using a Double antibody Radio Immuno Assay (DSL-4100 from Diagnostic System Laboratories Inc., USA).
The immature/prepubertal animals were of Chinese origin and purpose-bred as well. In total, 4 illustrative animals that were not sexually mature were chosen from the placebo group of a second toxicity study. They were approximately 3 years old and their body weights were 2.5–3.5 kg. All animals underwent a clinical inspection for ill health and were tested for tuberculosis and checked upon helminthes infections. Acclimatization of the animals was for a period of at least 4 weeks. Food consisted of a pelleted diet (Old World Monkey diet or a commercial pellet diet for primates), 120 gram/day for the sexually mature animals and 200 gram/day for the immature animals. They were fed additionally with fruits or vegetables and they were provided with tap water ad libitum. The animals were housed in climate-controlled cages (1–3 animals per cage) with a temperature of 19–25°C and a relative humidity of 30–70%.
After the experimental period, the animals were euthanized by an overdose of sodium pentobarbitone given intravenously. Animals were exsanguinated and the testes were fixed in Bouin’s fixative for at least 1 week, washed in alcohol 70%, processed and embedded in paraffin wax. Histological sections of about 4–5 μm in thickness were prepared and slides were stained either with haematoxylin and eosin (H&E) or with the periodic acid-Schiff-haematoxylin technique (PAS).
The studies were conducted in accordance with the requirements of current, internationally Good Laboratory Practice Standards. The in-life experimental procedures were subject to the provisions of the local Acts (Scientific Procedures of United Kingdom and Germany) on Animal Experimentation.
Results and Discussion
Spermatogenesis
Spermatogenesis occurs within the seminiferous tubules of the testis. It is a complex, dynamic process of continuous production of highly differentiated haploid spermatozoa from undifferentiated diploid stem cell spermatogonia. In this process, there are 3 important developmental phases, viz. the proliferative phase, the meiotic phase and spermiogenesis (Russell et al., 1990).
In the proliferative phase the spermatogonia (the primitive germ cells) undergo a series of mitotic divisions. The final mitotic division gives rise to the spermatocyte. The spermatogonia have been classified into two categories, distinguished by characteristic patterns of nuclear staining: the type A dense (Ad) and A pale (Ap). In the adult, the Ad spermatogonia are mitotically quiescent and are considered to be reserve stem cells. The Ap spermatogonia are mitotically active, renewing themselves and producing the first generation of differentiated spermatogonia, the B spermatogonia. The differentiation into these B spematogonia represents the crucial event of the initiation of spermatogenesis. Four types of B spermatogonia have been identified (B1, B2, B3, and B4), with smaller nuclei and increasing amounts of heterochromatin in each successive generation. These spermatogonia undergo sequential mitotic divisions resulting in the production of extraordinary numbers of spermatozoa that are characteristic of the macaque spermatogenesis (Marshall et al., 2005).
In the meiotic phase the spermatocytes are changing. The process begins with the duplication of the DNA during preleptotene. This first step in the meiotic phase is characterized by peripheral chromatin material in the nuclei. Full pairing and condensing of the chromosomes follows during pachytene, morphologically characterized by strands of chromatin separated by large clear areas within the progressively expanding nucleus. Finally, 2 reduction divisions gives rise to the spermatids.
Spermiogenesis covers the transformation of the immature spermatids into spermatozoa. It starts with simple round spermatids that undergo significant morphological changes. The nuclear DNA becomes highly condensed and elongates into a head region that is covered by a glycoprotein acrosome coat. The morphological detail of the developing acrosome is the basis of stage identification. This acrosome can only be visualized with a PAS stain that stains the glycoprotein component. The cytoplasm becomes a whip-like tail enclosing a flagellum and tightly packed mitochondria.
Within the seminiferous tubules, the various germ cells are arranged in a strict pattern. Germ cells are embedded tightly in between the Sertoli cells. The Sertoli cells regulate the development of the various germ cells and help them to move from their basal location up to the luminal side. In cross-sections of the tubules, as visible in routine sectioned slides, the spermatogonia are located closely to the basement membrane in between the Sertoli cells. Sertoli cells can be distinguished from germ cells because they have an elongated irregular-shaped nucleus with pale chromatin pattern, a single nucleolus and a significant amount of pale-staining cytoplasm. Sertoli cells are generally prismatic in shape and, although attached to the basement membrane, the cell length spans the full thickness of the germinative epithelium. The spermatocytes are situated more to the luminal side and the spermatids are present above them, near to the lumen of the tubule. The various cell types and their arrangements in the cross-sectioned tubule are depicted in Figure 1.
The Spermatogenic Cycle
The various generations of spermatogenic cells form characteristic stages (cell associations) within the seminiferous tubules. Despite a clear variation in the time of development and the morphology of the germ cells between various species, the organization of the different stages is based upon the same principle (Russell et al., 1990). In the cynomolgus and rhesus monkey, 12 morphological stages have been identified (Clermont and Leblond, 1959; Fouquet and Dadoune, 1986; Zhengwei et al., 1997). These 12 stages are associated with 14 steps in the development of the spermatid. The acrosome of the spermatids is present in all stages and is regarded as an important morphological marker. It forms a cap during steps 1–8 and carries on covering the elongating head during the later steps. The details of the acrosomic granules and cap formation are used as important morphological indicators for the early steps, whereas the shape of the head is used in the later steps.
The appearance of a new generation of spermatids from pachytene spermatocytes (step 1) is used to define the first morphological stage (Stage I). The production and division of secondary spermatocytes is defined as the last stage of the spermatogenic cycle (Stage XII). The duration of spermatogenesis in the cynomolgus monkey (from spermatogonium to fully developed elongated spermatid) is 42 days (Fouquet and Dadoune, 1986). The duration of one cycle of the seminiferous epithelium covering the 12 morphological stages is approximately 10.5 days (Fouquet and Dadoune, 1986; Aslam et al., 1999). The 12 different morphological stages, with the 14 developmental steps of the spermatids, are schematically drawn in Figure 2. In this figure also the duration of a specific stage (expressed as percentage of one cycle length) is given. In the next paragraph the characteristics of the different stages are discussed and illustrated by photomicrographs of testis sections (Figure 3).
Main Characteristics of the Cell Types and Stages
Spermatogonia
Spermatogonia are classified into types A and B based on their differentiation and developmental status.
Spermatogonia Type A
This cell type is located in close association with the basement membrane and is present in all stages but is few in number. The nucleus contains finely granular chromatin and 1 or 2 nucleoli. The Type A spermatogonia are subdivided into 2 types, dark (Adark) and pale (Apale) spermatogonia. This differentiation is based on the staining density of the nucleus, the nuclear size (approx. 6.4 μm and 8.4 μm, respectively) and upon the cytoplasmic content of PAS positive material. Typical examples are shown in Figures 1, 3A stage I and VI, 3B stage X and XI. The mitosis of Apale spermatogonia occur in stage VII, VIII, and IX (Fouquet and Dadouine, 1986).
Spermatogonia Type B
The B spermatogonia are the main progenitor cells. This cell type is characterized by a nucleus that contains a centrally located nucleolus and clumps or granules of darkly stained chromatin distributed along the nuclear membrane. Based on these characteristics, it can be differentiated from the type A spermatogonia (Figure 3A, stage VI). There are 4 generations of differentiated B spermatogonia (Marshall et al., 2005). They undergo a series of 4 mitotic divisions during one cycle: 1B1 → 2B2 → 4B3 → 8B4 → 16 spermatocytes. The mitosis of B1 occurs mainly in stage IX, that of B2 in stage XII, of B3 in stage II and B4 in stage IV. It is difficult to distinguish them from each other by routine staining procedures (Fouquet and Dadouine, 1986).
Spermatocytes
Spermatocytes are divided into the subtypes preleptotene, leptotene, and zygotene. The nuclei of the newly formed diploid preleptotene spermatocytes are indistinguishable from the B spermatogonia. However, when the chromosomes are duplicated during preleptotene to form a tetraploid cell, the nuclei of these spermatocytes are characterized by threadlike clumps of chromatin, which become progressively thicker and denser as they develop through meiotic prophase. (Examples are given in Figure 3B, stages VIII–XI).
Pachytene Spermatocytes
These cells develop from zygotene spermatocytes in stage XII. They are located closer to the tubular lumen than the other spermatocytes and contain a large round nucleus with chromatin organized in thick cords. The nucleolus is large and dark (Figure 3, stages I–XII). As the tetraploid cells progress through pachytene, the spermatocytes progressively expand in size. Following a very brief phase of diplotene and diakinesis, they undergo the first meiotic division to form diploid secondary spermatocytes and then rapidly enter the second meiotic division to form the haploid spermatids.
Spermatids
After the second division of secondary spematocytes in stage XII, the spermatids develop. The meiotic figures are specific for stage XII and easy recognizable (Figure 3B, stage XII). Morphological characteristics of the developing spermatids, defining the various stages, are described next and illustrated in Figure 3. The first 8 steps of spermatid development are recognized by acrosomic detail. The acrosome can only be visualized by PAS and even then, the structures such as the proacrosomal granule and early acrosomal cap are so small that they are difficult to observe in routine sections.
Stage I
Step 1 round spermatids lack distinguishing characteristics since proacrosomal granules are not visible. Step 13 elongated spermatids are also present in this stage and are flat, have a paddle-shaped nucleus and are partly covered by the acrosomic system (fused head cap and acrosome). They are fully condensed and dark. The apex becomes rounded and the spermatids are arranged in bundles within the seminiferous epithelium (Figure 3A, stage I). Step 13 is almost the last step in the development of the spermatids and is normally present in stages I to V (Figure 3A, stages I–V).
Stage II
Step 2 round spermatids show proacrosomal granules within proacrosomal vesicles that are formed initially during this stage. The small proacrosomal vesicles fuse to form one large vesicle containing a single fused granule. The vesicle touches the nucleus. Prominent Golgi complexes are noted as relative large intracytoplasmic eosinophilic structures. The step 13 elongated spermatids are still present but loose their bundle arrangement (Figure 3A, stage II).
Stage III
Step 3 round spermatids show an acrosomal vesicle that adhere to the nuclear membrane (Figure 3A, stage III). Step 13 spermatids are present.
Stage IV
Step 4 round spermatids show an acrosomal vesicle that flatten on the nuclear surface (Figure 3A, stage IV) forming a head cap-like structure. Step 13 elongated spermatids are present.
Stage V
In step 5 round spermatids, the acrosomal granule starts to spread over the nuclear membrane forming a small head cap (Figure 3A, stage V). In this stage the step 13 spermatids already start to pinch off parts of the cytoplasm, which are visible as small cellular remnants, the residual bodies (see also stages VI and VII).
Stage VI
In step 6 round spermatids, the acrosomal head cap covers one-fourth of the nuclear surface. Step 14 elongated spermatids can be seen in this stage. They represent the fully developed spermatids and are characteristically lined in a uniform row prior to sperm release from the testis. Residual bodies are a prominent feature in this stage (Figure 3A, stage VI).
Stage VII
In step 7 round spermatids, the acrosomal head cap extends to its maximum, covering one-third of the nuclear surface. The step 14 elongated spermatids are released into the lumen (spermiation). From this stage onwards only one generation of spermatids can be found. The residual bodies are still clearly visible at the border of the luminal space (Figure 3B, stage VII). They will either be extruded via the lumen or phagocytized by the Sertoli cells.
Stage VIII
In step 8 spermatids, the head cap is oriented to the basal membrane (Figure 3B, stage VIII). The spermatids are still round in this stage.
Stage IX
The nucleus in step 9 spermatids elongates and the part covered by head cap is conical giving it a peg top-like shape (Figure 3B, stage IX).
Stage X
The nuclei of step10 spermatids continues to elongate, the apex becomes clearly pointed (Figure 3B, stage X).
Stage XI
In step 11 spermatids the nucleus elongates even more and flattens considerably, giving it a shape of a paintbrush/paddle (Figure 3B, stage XI).
Stage XII
Nuclear flattening undergoes completion in step 12 spermatids. The apex becomes rounded (Figure 3B, stage XII). Meiosis of spermatocytes is characteristic for this stage (shown by meiotic figures), which is the initial process for step 1 spermatid formation.
Characteristics of Immature and Prepubertal Testes
The immature testis is characterized by the presence of only Sertoli cells and undifferentiated spermatogonia (Figure 4A). The diameter of cross-sectioned testis tubules is small, mainly because of a narrow tubular lumen. Testes weights are low (Simorangkir et al., 2005). The spermatogonia (Ad and Ap) are usually located on the basement membrane, but are sometimes found in the central area of the cord (Figure 4A). Up to the prepubertal period there is a slow increase in testicular weight and tubular diameter. In the (pre)pubertal testes there is a rapid growth and increase in tubular diameter. The first generation of B spermatogonia appears.
The sequential mitotic division of each B1 to B4 spermatogonium gives rise to 16 spermatocytes. With this spermatogonial mitotic activity spermatogenesis is initiated. The morphology of the testes in this period is characterized by high individual variation between animals of the same age. In some animals the testes show complete spermatogenesis up to mature spermatozoa and in others spermatogenesis is still incomplete. Even within one testis, a considerable variation can be present with areas of full spermatogenesis, areas with partial depletion of germ cells and areas with few germ cells.
When relatively young animals are used in studies, this variability in sexual maturity between individuals can be a complicating factor in the evaluation of the testis since histopathologic characteristics of immature and peripubertal testes may be indistinguishable from chemically induced toxicity. The morphological features of marked drug-induced testicular atrophy may closely resemble those of normal maturing testes. Severe drug-induced germ cell depletion, resulting in a negligible germ cell number, can mimic the immature testis. In peripubertal testes, spermatogenesis still can be suboptimal (lacking certain germ cell populations) and degenerating germ cells that slough into the tubular lumen can be a normal finding. Drug-induced hormonal disturbances however also can cause such a phenomenon, making judgment on the safety of the compound for the testis hard or even impossible. In dogs, similar difficulties have been described by Creasy et al. (2001).
Due to the small group sizes used in primate toxicity studies and the difficulties of assessing pubertal status at the start of the study, it is common that animals of different maturity are assigned to dose groups in a nonrandom manner. This makes differentiation of possible treatment-induced testicular toxicity from immature related absence or prepubertal related incomplete spermatogenesis at the end of the study very difficult. An example of such a case, a 4-week toxicity study with a mixture of immature/(peri)mature cynomolgus monkeys distributed among all groups, is given in Figure 4. In the immature animals of this study predosing testosterone levels were below 3.3 nmol/L and the diameters of the cross-sectioned fixed testes at the end of the study, varied between 0.9 and 1.1 cm. Testes of prepubertal animals showed incomplete spermatogenesis and predosing testosterone levels in these animals varied from 2.4 to 10.8 nmol/L. The diameters of these testes at the end of the study were between 1.4 and 1.9 cm. Sexually mature animals showed predosing testosterone levels between 8.8 and 90.5 nmol/L and testes diameters varied between 1.8 and 2.4 cm at the end of the study. Three animals had predosing testosterone levels below 15–20 nmol/L but at the end of the study, their testes diameters were between 1.8 and 2.2 cm. This indicated that these particular animals were prepubertal at the start of the study but attained maturity (with full spermatogenesis) at the end of the study.
To reduce these problems the monkeys used in toxicity studies should be of proven maturity. Full spermatogenesis, as judged morphologically by the presence of first young spermatozoa in the testis, is established between 3 years and 8 months and 4 years and 4 months (Dang and Meussy-Dessole, 1984). Other criteria of maturity are described by Vogel (2000) and comprise: (1) bodyweight >4.5 kg, (2) age: 4–5 years; testicular volume >10 ml (cross-section ~2.2 cm in diameter), (3) serum testosterone >15–20 nmol/L, and (4) proof of sperm in the ejaculate. Smedley et al. (2002) developed a statistical model to determine sexual maturity based on either age or body weight only.
In Figure 4B, an example is given of a testis of a prepubertal cynomolgus monkey. In the prepubertal testis both A and B spermatogonia are present. Besides the presence of spermatogonia, also spermiogenesis has already been started as is visible by the presence of pachytene spermatocytes and some round spermatids.
Immaturity and prepuberty can be predicted without first studying testis morphology on the basis of testosterone blood levels and testis diameter. Although other parameters are being used such as secondary sex characteristics, dentition and sperm counts, these are highly variable and therefore not very predictive for sexual maturity.
Concluding Remarks
The morphological characteristics of the different stages of the spermatogenic cycle in the cynomolgus monkey are well defined and most of them can be identified microscopically in routinely prepared H&E and PAS-stained sections. Since the binary decision key has been proven to be helpful for the determination of the different stages in the rat (Hess, 1990), we included a modified decision tree (Figure 5) that can be used for the classification of the different developmental stages of spermatogenesis in cynomolgus monkeys. Results obtained using this approach can help the pathologist in determining possible drug-related effects on the process of spermatogenesis and—in more detail—on the cellular level or stage(s) these effects takes place. This enables elucidation of the possible mechanisms of toxicity leading to the observed morphological changes. However, when relative young animals are used, this can be difficult or even impossible, since the morphology of immature or peripubertal testes can closely resemble drug-induced disturbances in the process of spermatogenesis.
The general process of spermatogenesis in the cynomolgus monkey is considered to be highly comparable to that in man. The regular morphology of the germinative epithelium in the cynomolgus monkey enables an even easier recognition of spermatogenic stages and abnormalities as compared to the human testis. Therefore, the cynomolgus monkey seems to be a suitable model to evaluate and predict potential toxicologic effects on human spermatogenesis. However, the main question remains whether compounds that disrupt spermatogenesis or induce other changes in the cynomolgus monkey testes, will do the same in humans. Unfortunately, the literature concerning such cases is lacking.
Footnotes
Acknowledgments
The authors would like to thank Mrs. S. van den Wijngaard for her excellent histotechnical help.