Correspondence: Joji Yuasa MD, Department of Urology, Chiba University School of Medicine, Chiba 260–8670, Japan
Abstract Background: Little is known about the morphological aspects of the postnatal development of the testis in Japanese children. By investigating the normal development of the testis, clinicians in urology can give better strategies to their patients. In addition, the pattern of development will improve the understanding of the effect of xenoestrogens.
Methods: Biopsied testis samples of 68 patients with undescended testes aged between 2 and 21 years were examined by light microscopy. Nineteen patients with normal histology of the bilateral testes were selected for this study. For light microscopy, paraffin sections were stained with hematoxylin and eosin. In addition, using electron microscope, Epon sections stained with lead citrate and uranyl acetate were observed. Semi-thin sections stained with toluidine blue were also used for light microscopy.
Results: Gonocytes were observed in the testis of the 2-year-old boys. Spermatocytes developed by 4 years of age and spermatids developed by 11 years of age. The immature Sertoli–Sertoli ectoplasmic junction was observed as early as 4 years of age, and it was completed by 9 years of age. Mature myoid cells were observed by the age of 13 years. Immature Leydig cells were found at 7 years of age and the cells matured by 13 years of age.
Conclusion: A pattern of postnatal development of the testis in Japanese children was presented.
A few reports have appeared on the postnatal development of the human testis.1–3 The present study will show the development of the testis in Japanese children, giving a guideline to diagnosis and therapy in relation to testicular diseases, such as, undescended testes, acute lymphatic leukemia (ALL) and torsion of the spermatic cord. Since Carlsen et al. reported that semen quality in humans has declined in the past five decades,4 it has been a subject of controversy. Some skeptics point out that data from several decades ago are unreliable. However, the influence of xenoestrogens on the male reproductive system has been strongly implicated in this decline.5 This study will also show a standard pattern of postnatal development of the testis in the Japanese child and will contribute to future studies on the influence of endocrine disruptors, such as, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and polychlorinated biphenyls (PCB).
Biopsy samples were obtained from the testes of 68 patients complaining of undescended testes after obtaining the patient’s and/or his parents’ consent. The biopsied samples were obtained at the time of orchiopexy for the purpose of their prognostic value with respect to fertility. They were divided into two parts with razor blades. One part was fixed in Bouin’s fixative and embedded in paraffin wax for light microscopy and the other was fixed in 3% glutaraldehyde and embedded in Epon for electron microscopy. The specimens had been stored in our hospitals. At the time of the biopsies the patients ranged in age from 2 to 21 years.
Nineteen cases showing normal histology by age were selected for the present study. These cases included patients of 2, 3, 4, 5, 6, 7, 9, 11, 13 and 21 years of age. Since 77% of descended testes from patients with unilateral cryptorchidism did not show any lesion,6 a biopsied sample showing the most differentiated germ cells in each age group was regarded as normal in the present study.
The development of spermatids was divided into eight steps according to Holstein7 and Holstein and Roosen-Runge.8
Postnatal development of spermatogenic cells
Gonocytes were recognized in the testis of the 2-year-old boy. The gonocytes were scattered among the Sertoli cells. The cytoplasm was paler than that of the Sertoli cell. The size of the nucleus was larger than that of the Sertoli cell. At the age of 3 years spermatogonia were observed on the basal lamina. The cytoplasm was paler than that of the Sertoli cell and contained fewer cell organelles. The long axis of the oval nucleus was parallel to the basal lamina. At the age of 4–7 years, preleptotene spermatocytes were observed (Fig. 1). Crystalloid of Lubarsch was also observed in the spermatogonia (Fig. 2). Cap phase, or step 4 spermatids, were recognized in the testis of the 11-year-old boy (Fig. 3). Mature, or step 8 spermatids, were recognized in the testis of the 13-year-old boy (Fig. 4). The postnatal development of the germ cells obtained from the present study is summarized in Fig. 5.
Formation of the Sertoli–Sertoli ectoplasmic specialization
The testes of patients younger than 4 years showed no specific specialization between Sertoli cells. Primitive ectoplasmic specializations between the Sertoli cells were observed in the seminiferous epithelium of the 4-year-old boy (Fig. 2). The primitive specialization consisted of the cell membrane of the Sertoli cell with several tight junctions and a fragmented layer of a subsurface cistern of the endoplasmic reticulum. Actin filaments between the cell membrane and the subsurface cistern were not fully developed.9 The ectoplasmic specialization was completed by 9 years of age (Fig. 6).
Development of the interstitial tissue of the testis
Leydig cells were not recognized in the testes of 2–6-year-olds. Only fibroblast-like cells were observed in the interstitial tissue. At the age of 7 years, immature Leydig cells were recognized in the interstitial tissue. In the immature Leydig cells, some of the mitochondria contained tubular cristae. Developed smooth endoplasmic reticulum was also observed. The immature Leydig cells were also observed in the testis of the 9-year-old boy. Mature Leydig cells were observed in the testis of the 13-year-old boy (Fig. 7). The Leydig cell was characterized by mitochondria with tubular cristae and fully developed smooth endoplasmic reticulum in the cytoplasm. Light microscopy showed Reinke’s crystals in 21-year-old to 36-year-old patients with Sertoli cell only syndrome.
The development of immature myoid cells was recognized in the testis of the 7-year-old boy. The cells were flat in shape and closely faced to the seminiferous tubule. In the cytoplasm, scattered bundles of actin filaments10 were observed in addition to 10 nm filaments. The immature myoid cells with more actin filaments were also observed in the testis of the 13-year-old boy (Fig. 8).
There have been a few studies on the development of the testis in Japanese boys.1,3 Results of the present study provide additional data on the development of the testis.
According to the light microscopy findings by Sasaki,1 spermatocytes appeared at 10 years of age and spermatids appeared at 11 years of age. Our results showed that spermatocytes appeared at 4 years, step 4 spermatids at 11 years and step 8 spermatids at 13 years of age. Since spermatozoa appeared at 14 years of age in Sasaki’s study and the present study showed step 8 spermatids at 13 years of age, it is reasonable to assume that the 10- and 11-year-old boys in Sasaki’s study were retarded in their testis development. This is supported by the fact that spermatocytes appeared at 4 years of age in German boys.2 The crystalloid of Lubarsch was observed in the spermatogonia of a 4-year-old boy in the present study. This does not mean that the crystalloid appears at this age, since Seguchi and Hadziselimovic reported that the crystalloids were observed in patients as young as 4 months.11
As to the development of the ectoplasmic specialization between Sertoli cells, the results of the present study showed that the immature ones appeared at 4 years of age and that the specialization was completed by 9 years of age. This corresponds with the results of previous reports.3,12,13 After completion of the specialization, secondary spermatocytes and spermatids differentiate.14,15
The Reinke’s crystal was reported to appear at 13 years of age. The crystal was not found in the present study, even in the testes of a 21-year-old man, because the biopsied specimen covered only a limited area of the testis.
Recently, urologists have tended to avoid testicular biopsy. Since there has been no standard for the postnatal development of the testis in the Japanese child, there is now a good opportunity to establish a standard. The developing pattern shown in the present study gives a guideline to diagnosis for conditions, such as, undescended testes, varicocele, or testicular tumors. It also provides information on the harmful influence of anticancer chemicals. Recently, influences by environmental hormones, or xenoestrogens, were suggested. A standard for the postnatal development of the human testis should be established so that we can determine the influence of xenoestrogens on the human testis.