Asymmetric Distribution of UCH‐L1 in Spermatogonia Is Associated With Maintenance and Differentiation of Spermatogonial Stem Cells

Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, direct evidence for asymmetric division of mammalian spermatogonial stem cells (SSCs) has been scarce. Here, we report that ubiquitin carboxy‐terminal hydrolase 1 (UCH‐L1) is expressed in type A (A_s, A_pr, and A_al) spermatogonia located at the basement membrane (BM) of seminiferous tubules at high and low levels, but not in differentiated germ cells distant from the BM. Asymmetric segregation of UCH‐L1 was associated with self‐renewal versus differentiation divisions of SSCs as defined by co‐localization of UCH‐L1^high and PLZF, a known determinant of undifferentiated SSCs, versus co‐localization of UCH‐L1^low/? with proteins expressed during SSC differentiation (DAZL, DDX4, c‐KIT). In vitro, gonocytes/spermatogonia frequently underwent asymmetric divisions characterized by unequal segregation of UCH‐L1 and PLZF. Importantly, we could also demonstrate asymmetric segregation of UCH‐L1 and PLZF in situ in seminiferous tubules. Expression level of UCH‐L1 in the immature testis where spermatogenesis was not complete was not affected by the location of germ cells relative to the BM, whereas UCH‐L1‐positive spermatogonia were exclusively located at the BM in the adult testis. Asymmetric division of SSCs appeared to be affected by interaction with supporting somatic cells and extracelluar matrix. These findings for the first time provide direct evidence for existence of asymmetric division during SSCs self‐renewal and differentiation in mammalian spermatogenesis. J. Cell. Physiol. 220: 460–468, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation

Although methods to assess testis cell populations are established in mice, the detailed validation of similar methods for bovine testis cells is necessary for the development of emerging technologies such as male germ cell transplantation. As young calves provide donor cells for germ cell transplantation, we characterized cell populations from three key pre-pubertal stages. Nine Angus bull calves were selected to represent three stages of testis development at ages (and testis weights) of 2–3 months (Stage 1, 10 g), 4–5 months (Stage 2, 35 g), and 6–7 months (Stage 3, 70 g). The proportion and absolute numbers of germ and somatic cells in fixed sections and from enzymatically dissociated seminiferous tubules were assessed. Germ cells were identified by DBA and PGP9.5 staining, and Sertoli cells by vimentin and GATA-4 staining. Staining of serial sections confirmed that DBA and PGP9.5 identified similar cells, which were complementary to those stained for vimentin and GATA-4. In fixed tubules, the proportion of cells within tubules that were positive for DBA and PGP9.5 increased nearly three-fold from Stage 1 to Stage 2 with no further increase at Stage 3. Absolute numbers of spermatogonia also increased between Stages 1 and 2. After enzymatic dissociation of tubules, three times more DBA- and PGP9.5-positive cells were isolated from Stage 3 testes than from either Stage 1 or 2 testes. A higher proportion of spermatogonia was observed after enzymatic isolation than were present in seminiferous tubules. These data should help to predict the yield and expected proportions of spermatogonia from three distinct stages of testis development in pre-pubertal bull calves.     

Spermatogonial stem cell markers and niche in equids

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and are located in a highly dynamic microenvironment called "niche" that influences all aspects of stem cell function, including homing, self-renewal and differentiation. Several studies have recently identified specific proteins that regulate the fate of SSCs. These studies also aimed at identifying surface markers that would facilitate the isolation of these cells in different vertebrate species. The present study is the first to investigate SSC physiology and niche in stallions and to offer a comparative evaluation of undifferentiated type A spermatogonia (Aund) markers (GFRA1, PLZF and CSF1R) in three different domestic equid species (stallions, donkeys, and mules). Aund were first characterized according to their morphology and expression of the GFRA1 receptor. Our findings strongly suggest that in stallions these cells were preferentially located in the areas facing the interstitium, particularly those nearby blood vessels. This distribution is similar to what has been observed in other vertebrate species. In addition, all three Aund markers were expressed in the equid species evaluated in this study. These markers have been well characterized in other mammalian species, which suggests that the molecular mechanisms that maintain the niche and Aund/SSCs physiology are conserved among mammals. We hope that our findings will help future studies needing isolation and cryopreservation of equids SSCs. In addition, our data will be very useful for studies that aim at preserving the germplasm of valuable animals, and involve germ cell transplantation or xenografts of equids testis fragments/germ cells suspensions.     

A synergistic effect of insulin-like growth factor (IGF-I) on equine luteinizing hormone (eLH)-induced testosterone production from cultured Leydig cells of horses

Localization of IGF-I and IGF-IR were observed in Leydig cells of horses using immunohistochemistry (IHC), suggesting IGF-I may play a role in equine Leydig cell steroidogenesis. Previous studies in other species have indicated that IGF-I increases basal and/or LH/hCG-induced testosterone production. The objectives of this study were to (1) test the synergistic effect of IGF-I on eLH-induced testosterone production in cultured equine Leydig cells and (2) determine if this effect is reproductive stage-dependent. Testes were collected from five pubertal (1.1±0.1 year; 1-1.5 year) and eight post-pubertal (2.88±0.35 years; 2-4 years) stallions during routine castrations at the UC Davis Veterinary Hospital. Leydig cells were isolated using validated enzymatic and mechanical procedures. Leydig cells were treated without (control) or with increasing concentrations of purified pituitary-derived eLH and/or recombinant human IGF-I (rhIGF-I) and incubated under 95% air: 5% CO(2) at 32°C for 24h. After 24h, culture media was collected and frozen at -20°C until analyzed for testosterone by a validated radioimmunoassay (RIA). In pubertal stallions, treatment with both increasing concentrations of rhIGF-I and 5ng/ml of eLH failed to demonstrate a significant difference in testosterone production compared with 5ng/ml of eLH only. However, in post-pubertal stallions, a significant increase in the concentration of testosterone in culture media was observed from Leydig cells treated with various concentrations of rhIGF-I and 1 or 5ng/ml of eLH compared with 1 or 5ng/ml of eLH only. In conclusion, IGF-I has a synergistic effect on eLH-induced testosterone production in cultured equine Leydig cells from post-pubertal but not pubertal stallions.     

Lactoferrin expression and secretion in the stallion epididymis

Lactoferrin is one of the most abundant proteins secreted by the stallion epididymis, but its cellular localization and regulation remain unknown. This study was designed to address the following objectives: (1) identify the epididymal cell types producing lactoferrin in pre-pubertal, peri-pubertal and post-pubertal animals; (2) demonstrate that lactoferrin binds to stallion sperm; and (3) determine if testosterone and estradiol regulate lactoferrin secretion in vitro. Using an immunohistochemical method, lactoferrin was localized in the cytoplasm of principal cells in the corpus and cauda of peri- and post-pubertal animals. The epididymis of pre-pubertal animals did not express lactoferrin. Immunolabeling of lactoferrin was also observed on the mid-piece and tail of the sperm. The role of estradiol and testosterone in regulating secretion of lactoferrin in the post-pubertal epididymis was investigated using tissue culture methods. Lactoferrin concentration in the culture media was determined by validated enzyme-linked immunosorbent assays (ELISA). Testosterone did not increase the concentration of lactoferrin in the media in any epididymal region. In contrast, estradiol-17β significantly increased the concentration of lactoferrin in the media containing tissue from the cauda. In conclusion, the expression of lactoferrin was found in the cytoplasm of principal cells in the corpus and cauda of the epididymis in peri- and post-pubertal stallions but not pre-pubertal stallions. Furthermore, lactoferrin binds to sperm, suggesting a biological role for protection or regulation of sperm in the corpus and cauda. In addition, estrogen appears to regulate lactoferrin secretion in the cauda of the epididymis in post-pubertal stallions.     

A quantitative study of Sertoli cell and germ cell populations as related to sexual development and aging in the stallion

Testes from 47 stallions, 1-20 yr of age, were used to examine the influence of age on Sertoli and germ cell populations as well as on functional activity of Sertoli cells. For these stallions, the number of Sertoli cells per paired testes declined linearly with age, and was only 41.7% as great at age 20 as at age 2. However, development of reproductive organs proceeded until age 12-13, as evident from increases in paired testes weight and quantitative rates of spermatozoal production. Although the absolute number of Sertoli cells declined during this period of development, individual Sertoli cells displayed a remarkable capacity to accommodate greater numbers of developing germ cells. Between age 2 and age 12, the mean numbers of developing spermatogonia, young primary spermatocytes, old primary spermatocytes, and round spermatids supported by each Sertoli cell at Stage I of spermatogenesis increased by 49, 176, 153, and 161%, respectively.     

Essential role of mouse Dead end1 in the maintenance of spermatogonia

Dead end is a vertebrate-specific RNA-binding protein implicated in germ cell development. We have previously shown that mouse Dead end1 (DND1) is expressed in male embryonic germ cells and directly interacts with NANOS2 to cooperatively promote sexual differentiation of fetal germ cells. In addition, we have also reported that NANOS2 is expressed in self-renewing spermatogonial stem cells and is required for the maintenance of the stem cell state. However, it remains to be determined whether DND1 works with NANOS2 in the spermatogonia. Here, we show that DND1 is expressed in a subpopulation of differentiating spermatogonia and undifferentiated spermatogonia, including NANOS2-positive spermatogonia. Conditional disruption of DND1 depleted both differentiating and undifferentiated spermatogonia; however, the numbers of A_single and A_paired spermatogonia were preferentially decreased as compared with those of A_aligned spermatogonia. Finally, we found that postnatal DND1 associates with NANOS2 in vivo, independently of RNA, and interacts with some of NANOS2-target mRNAs. These data not only suggest that DND1 is a partner of NANOS2 in undifferentiated spermatogonia as well as in male embryonic germ cells, but also show that DND1 plays an essential role in the survival of differentiating spermatogonia.     

Immunohistochemical distribution of sulfhydryl oxidase in the human testis

Summary Sulfhydryl oxidase (SOx) is an enzyme that catalyzes the oxidation of sulfhydryl compounds. It is present in mitochondria of certain testicular cells at specific stages of functional activation. In the mature human testis moderate SOx immunoreactivity is found in Leydig cells, and lacking in Sertoli and in peritubular cells. The A_dark spermatogonia usually contain immuno-reactive mitochondria, while in A_pale spermatogonia immunoreactivity is mostly low. In stage V of spermatogenesis, A_pale spermatogonia were found containing immunoreactive material. Leptotene (stages IV and V) and zygotene (stage VI) primary spermatocytes display a moderate immunoreaction. It is strongest in pachytene spermatocytes of stages I–IV, decreases in stage V, and is low during diakinesis and in secondary spermatocytes. Late spermatids usually show a stronger immunoreactivity than early spermatids. At stage V of spermatogenesis the late spermatids contain only few immunoreactive particles. Spermatozoa are free of SOx-immunoreactive mitochondria. In residual bodies small amounts of SOx-immunoreactive particles are seen. Compared to rat and hamster testis, SOx immunoreactivity of the human testis is less clearly stage-dependent and it is not confined to certain germ cell stages. As deduced from the findings in patients with spermatogenic disorders, the SOx immunoreactivity of spermatogonia in human testis seems to be of diagnostic relevance.     

Immunolocalization of estrogen receptor alpha, estrogen receptor beta and androgen receptor in the pre-, peri- and post-pubertal stallion testis

In various species, androgens and estrogens regulate the function of testicular Leydig, Sertoli, peritubular myoid, and germ cells by binding to their respective receptors and eliciting a cellular response. Androgen receptor (AR) is expressed in Sertoli cells, peritubular myoid cells, Leydig cells and perivascular smooth muscle cells in the testis depending on the species, but its presence in germ cells remains controversial. Two different estrogen receptors have been identified, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), and their localization and function in testicular cells varies depending on the species, developmental stage of the cell and type of receptor. The localization of AR in an immature and mature stallion has been reported but estrogen receptors have only been reported for the mature stallion. In the present study, the localizations of AR and ERα/ERβ were investigated in pre-pubertal, peri-pubertal and post-pubertal stallions. Testes were collected by routine castration from 21 horses, of light horse breeds (3 months-27 years). Animals were divided into the following age groups: pre-pubertal (3-11 months; n=7), peri-pubertal (12-23 months; n=7) and post-pubertal (2-27 years; n=7). Testicular tissue samples were fixed and embedded, and the presence of AR, ERα and ERβ was investigated by immunohistochemistry (IHC) using procedures previously validated for the horse. Primary antibodies used were rabbit anti-human AR, mouse anti-human ERβ and rabbit anti-mouse ERα. Sections of each region were incubated with normal rabbit serum (NRS; AR and ERα) or mouse IgG (ERβ) instead of primary antibody to generate negative controls. Androgen receptors were localized in Leydig, Sertoli and peritubular myoid cells of all ages. Estrogen receptor alpha was localized in Leydig and germ cells of all ages but only in pre- and peri-pubertal Sertoli cells and post-pubertal peritubular myoid cells. Estrogen receptor beta was localized in Leydig and Sertoli cells of all ages but in only pre-pubertal germ cells and absent in peritubular myoid cells of all ages. Taken together, the data suggest that estrogen regulates steroidogenesis by acting through ERα and ERβ in the Leydig cells and promotes gametogenesis by acting through ERβ in the Sertoli cells and ERα in the germ cells. In contrast androgen receptors are not found in germ cells throughout development and thus are likely to support spermatogenesis by way of a paracrine/autocrine pathway via its receptors in Leydig, Sertoli and peritubular myoid cells.     

The heterogeneity of spermatogonia is revealed by their topology and expression of marker proteins including the germ cell-specific proteins Nanos2 and Nanos3

Spermatogonial stem cells (SSCs) reside in undifferentiated type-A spermatogonia and contribute to continuous spermatogenesis by maintaining the balance between self-renewal and differentiation, thereby meeting the biological demand in the testis. Spermatogonia have to date been characterized principally through their morphology, but we herein report the detailed characterization of undifferentiated spermatogonia in mouse testes based on their gene expression profiles in combination with topological features. The detection of the germ cell-specific proteins Nanos2 and Nanos3 as markers of spermatogonia has enabled the clear dissection of complex populations of these cells as Nanos2 was recently shown to be involved in the maintenance of stem cells. Nanos2 is found to be almost exclusively expressed in A_s to A_pr cells, whereas Nanos3 is detectable in most undifferentiated spermatogonia (A_s to A_al) and differentiating A₁ spermatogonia. In our present study, we find that A_s and A_pr can be basically classified into three categories: (1) GFRα1⁺Nanos2⁺Nanos3⁻Ngn3⁻, (2) GFRα1⁺Nanos2⁺Nanos3⁺Ngn3⁻, and (3) GFRα1⁻Nanos2 ^± Nanos3⁺Ngn3⁺. We propose that the first of these groups is most likely to include the stem cell population and that Nanos3 may function in transit amplifying cells.     

Phenotypic Plasticity of Mouse Spermatogonial Stem Cells

Background

Spermatogonial stem cells (SSCs) continuously undergo self-renewal division to support spermatogenesis. SSCs are thought to have a fixed phenotype, and development of a germ cell transplantation technique facilitated their characterization and prospective isolation in a deterministic manner; however, our in vitro SSC culture experiments indicated heterogeneity of cultured cells and suggested that they might not follow deterministic fate commitment in vitro.

Methodology and Principal Findings

In this study, we report phenotypic plasticity of SSCs. Although c-kit tyrosine kinase receptor (Kit) is not expressed in SSCs in vivo, it was upregulated when SSCs were cultured on laminin in vitro. Both Kit⁻ and Kit⁺ cells in culture showed comparable levels of SSC activity after germ cell transplantation. Unlike differentiating spermatogonia that depend on Kit for survival and proliferation, Kit expressed on SSCs did not play any role in SSC self-renewal. Moreover, Kit expression on SSCs changed dynamically once proliferation began after germ cell transplantation in vivo.

Conclusions/Significance

These results indicate that SSCs can change their phenotype according to their microenvironment and stochastically express Kit. Our results also suggest that activated and non-activated SSCs show distinct phenotypes.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

The Transcriptional Cell Atlas of Testis Development in Sheep at Pre-Sexual Maturity

Sheep testes undergo a dramatic rate of development with structural changes during pre-sexual maturity, including the proliferation and maturation of somatic niche cells and the initiation of spermatogenesis. To explore this complex process, 12,843 testicular cells from three males at pre-sexual maturity (three-month-old) were sequenced using the 10× Genomics Chromium^TM single-cell RNA-seq (scRNA-seq) technology. Nine testicular somatic cell types (Sertoli cells, myoid cells, monocytes, macrophages, Leydig cells, dendritic cells, endothelial cells, smooth muscle cells, and leukocytes) and an unknown cell cluster were observed. In particular, five male germ cell types (including two types of undifferentiated spermatogonia (A_pale and A_dark), primary spermatocytes, secondary spermatocytes, and sperm cells) were identified. Interestingly, A_pale and A_dark were found to be two distinct states of undifferentiated spermatogonia. Further analysis identified specific marker genes, including UCHL1, DDX4, SOHLH1, KITLG, and PCNA, in the germ cells at different states of differentiation. The study revealed significant changes in germline stem cells at pre-sexual maturation, paving the way to explore the candidate factors and pathways for the regulation of germ and somatic cells, and to provide us with opportunities for the establishment of livestock stem cell breeding programs.     

Expansion and long-term culture of human spermatogonial stem cells via the activation of SMAD3 and AKT pathways

Spermatogonial stem cells (SSCs) can differentiate into spermatids, reflecting that they could be used in reproductive medicine for treating male infertility. SSCs are able to become embryonic stem-like cells with the potentials of differentiating into numerous cell types of the three germ layers and they can transdifferentiate to mature and functional cells of other lineages, highlighting significant applications of human SSCs for treating human diseases. However, human SSCs are very rare and a long-term culture system of human SSCs has not yet established. This aim of study was to isolate, identify and culture human SSCs for a long period. We isolated GPR125-positive spermatogonia with high purity and viability from adult human testicular tissues utilizing the two-step enzymatic digestion and magnetic-activated cell sorting with antibody against GPR125. These freshly isolated cells expressed a number of markers for SSCs, including GPR125, PLZF, GFRA1, RET, THY1, UCHL1 and MAGEA4, but not the hallmarks for spermatocytes and spermatozoa, e.g. SYCP1, SYCP3, PRM1, and TNP1. The isolated human SSCs could be cultured for two months with a significant increase of cell number with the defined medium containing growth factors and hydrogel. Notably, the expression of numerous SSC markers was maintained during the cultivation of human SSCs. Furthermore, SMAD3 and AKT phosphorylation was enhanced during the culture of human SSCs. Collectively, these results suggest that human SSCs can be cultivated for a long period and expanded whilst retaining an undifferentiated status via the activation of SMAD3 and AKT pathways. This study could provide sufficient cells of SSCs for their basic research and clinic applications in reproductive and regenerative medicine.     

The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse

Background  

Life-long production of spermatozoa depends on spermatogonial stem cells. Spermatogonial stem cells exist among the most primitive population of germ cells – undifferentiated spermatogonia. Transplantation experiments have demonstrated the functional heterogeneity of undifferentiated spermatogonia. Although the undifferentiated spermatogonia can be topographically divided into A_s (single), A_pr (paired), and A_al (aligned) spermatogonia, subdivision of this primitive cell population using cytological markers would greatly facilitate characterization of their functions.     

Morphological and histochemical characterization of the seminiferous epithelial and Leydig cells of the turkey

Unlike mammals, there is little fundamental information about spermatogenesis in birds. This study was undertaken to clarify the morphology, histochemistry, and lectin affinity of the seminiferous epithelial cells and Leydig cells in pre-pubertal (8- to 15-week old) and adult (40- to 44-week old) domestic turkeys. In adult turkeys, three types of spermatogonia were defined based on their chromatin distribution and nuclear morphology: the dark type A (A(d)); the pale type A (A(p)); and the type B. The A(d) is the least numerous and least conspicuous and consequently difficult to locate. Based on its spatial distribution and overall morphology, type A(d) spermatogonia were postulated to be the spermatogonia stem cells in the turkey. Antibodies to c-kit were localized to spermatogonia in the pre-pubertal and to a lesser extent in adult males. Peanut agglutinin (PNA) was specific for spermatocytes in the pre-pubertal males and spermatogonia and early spermatocytes in adult males. Wheat-germ agglutinin (WGA) highlighted Sertoli cells in both age groups. Bandeiraea simplicifolia I, soybean agglutinin, and winged-pea agglutinin staining were limited to the wall of the seminiferous tubule and some extra-tubular cell types. Concanavalin A staining was diffuse and not cell-specific and, therefore, could not be used to selectively identify a particular cell type. It was concluded that WGA and PNA could aid in identifying specific cell types in the seminiferous epithelium of testis from pre-pubertal and mature turkeys. Only Leydig cells were alkaline phosphatase reactive in the mature turkey testes. The information from this study is being used to adapt techniques for the isolation and partial purification developed for mammalian spermatogonia to avian spermatogonia and other specific cell types in the testes.     

Increased daily sperm production in the breeding season of stallions is explained by an elevated population of spermatogonia

Seasonal variation in number of spermatogonia and germ cell degeneration was evaluated to determine which mechanism might explain seasonal differences in daily sperm production per testis (DSP/testis) or per g parenchyma (DSP/g) in stallions. Comparing 28 adult stallions (4 to 20 yr old) in each of the nonbreeding (December-January) and breeding (June-July) seasons, the population of type A spermatogonia was more than two times greater (P less than 0.01) in the breeding season. While the number of type B spermatogonia also was elevated (P less than 0.01) in the breeding season, the number of type B spermatogonia/type A spermatogonium was similar (P greater than 0.05) between seasons. Daily sperm production/testis based on each cell type from type B spermatogonia to spermatids with elongated nuclei was lower (P less than 0.01) in the nonbreeding season. Based on DSP/g, there was significant degeneration during the meiotic divisions in the nonbreeding season. However, this reduction in potential spermatozoan production was not significant (P greater than 0.05) when considering DSP/testis. Significant germ cell degeneration also occurred in the breeding season between type B spermatogonia and primary spermatocytes. However, the type A spermatogonial population was sufficiently elevated to override this degeneration and to explain elevated production of sperm in the breeding season of stallions.     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.