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Testis |
MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, Edinburgh EH16 4SB, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gamete biology, male reproductive tract, Sertoli cells, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In species with a short gestation, development of fetal germ cells occurs in a synchronous manner. For example, in mice, PGCs within the testis cords differentiate into a single population of fetal germ cells, usually referred to as "gonocytes," that actively proliferate until about Day 15.5 p.c. and then remain arrested in G1 until Day 1.5 postpartum, when they resume mitotic activity [7, 8]. Two days before they resume reproliferation, the gonocytes relocate to the periphery of the cord, make contact with the basement membrane, and thereafter, undergo further differentiation via a process that is as yet poorly understood [9].
In humans, the fetal germ cell population in the testis is variously referred to as consisting of "gonocytes" or "prespermatogonia" and is often assumed to represent a homogenous population, as in rodents. This view is supported by the expression of proteins such as DAZL [10], VASA [11], and CHK2 [12], which are reported to be present in all germ cells during the second trimester. However, histological evaluations conducted using electron microscopy more than 20 yr ago recorded the existence of distinct subpopulations of human fetal germ cells based on their morphological appearance [2, 13–15]. Wartenberg [2, 15] classified the fetal germ cells into different subpopulations, these being gonocytes (i.e., the PGC population after it was resident in testis cords) and prospermatogonia. Wartenberg divided the latter into M prospermatogonia (mitotically active), T1 prospermatogonia (nondividing/resting), and T2 prospermatogonia (those that had resumed mitotic activity). Holstein et al. [14] identified a germ cell type in 8-, 10-, and 13-wk embryos that had a large nucleus and a prominent nucleolus that they called "gonocytes." In more mature fetuses (13 and 22 wk), those authors noted that some germ cells had features distinct from the gonocytes and suggested that these be classified as "fetal spermatogonia." Fukuda et al. [13] examined fetuses between 9 and 30 wk of gestation and reported that they were able to identify three morphologically distinct germ cell types, which they called gonocytes, intermediate cells, and fetal spermatogonia.
The aims of the present study were to evaluate the patterns of protein expression in testes from the first and second trimesters, to determine whether one or more populations of germ cells could be identified in paraffin-fixed material by means of immunohistochemical evaluation, and to evaluate whether the pattern of protein expression was correlated with changes in cell morphology and/or differentiation status of germ cells. Germ cells were identified using antibodies directed against proteins reported to be expressed in human fetal germ cells or testicular germ cell tumors [12, 16, 17]. These proteins included those known to be expressed in PGCs, such as placental alkaline phosphatase (PLAP) [2], C-KIT (the membrane tyrosine kinase receptor for the kit ligand [18]), and OCT4, which is a marker of pluripotency [19]. Other proteins were selected because they are believed to be involved in regulation of the cell cycle (e.g., CHK2, [20] and PCTAIRE-1 [21]). The pattern of expression of MAGE-A4 (melanoma antigen A4) was of particular interest, because this protein is a member of a testis-cancer family of antigens and because, in the adult testis, intense immunostaining for MAGE-A4 has been detected in the nuclei and cytoplasm of spermatogonia [22].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human fetal testes were obtained following termination of pregnancy during the first trimester (7.7–9.2 wk [54–65 days], n =10) and second trimester (14–19 wk, n = 15). Women gave consent according to national guidelines [23], and the present study was approved by the Lothian Pediatrics/Reproductive Medicine Research Ethics subcommittee. Termination of pregnancy was induced by treatment with mifepristone (200 mg orally) followed by misoprostol (800 mg every 3 h per vaginam; Pharmacia, Milton Keynes, U.K.). None of the terminations were for reasons of fetal abnormality, and all fetuses appeared to be morphologically normal. Gestational age was determined by ultrasound and confirmed by subsequent measurement of foot length for the second-trimester samples. Testes were fixed in Bouin fluid for 5 h followed by transfer to 70% ethanol and processed into paraffin wax using standard methods. Sections from all testes were stained with hematoxylin-and-eosin using standard methods to check for tissue integrity and to provide an overall assessment of morphological development before analysis by immunohistochemistry.
The sex of the first-trimester samples was determined by polymerase chain reaction (PCR) analysis of genomic DNA using primers directed against the SRY gene. Briefly, 100 µl of 25 mM NaOH/0.2 M EDTA were added to approximately 50 mg of tissue and heated at 95°C for 20 min before the addition of 100 µl of 40 mM Tris-HCl. The samples were then mixed by vortex and centrifuged briefly. Five microliters of supernatant were used in a standard 25-µl PCR reaction with Qiagen HotStar Taq. The SRY primers used were those detailed by Friel et al. [24]: forward, 5'-ACAGTAAAGGCAACGTCCAG, and reverse, 5'-ATCTGCGGGAAGCAAACTGC].
Immunohistochemistry
Details of all antibodies used in the present study and the dilutions used for immunohistochemistry with a single antibody are shown in Table 1. Sections (thickness, 5 µm) of Bouin-fixed, paraffin-embedded fetal testes were mounted on charged glass slides (BDH Chemicals, Poole, U.K.), dewaxed, rehydrated, and subjected to heat-induced antigen retrieval in 0.1 M citrate (pH 6) if required (Table 1). Endogenous peroxidase activity was blocked by incubation in 3% (v/v) H2O2 in methanol for 30 min. After a wash in water, slides were transferred into Tris-buffered saline (TBS; 0.05 M Tris and 0.85% NaCl, pH 7.6). Endogenous biotin was blocked using an avidin/biotin blocking kit (Vector Laboratories, Inc., Peterborough, U.K.) according to manufacturer's instructions but incorporating a protein block of 5% BSA (Sigma) in TBS in the avidin step. Antibodies were diluted in 5% BSA/TBS (Table 1) and applied to the sections at 4°C overnight in a humidified chamber.
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3,3-Diaminobenzidine Tetrahydrochloride Visualization
Sections were washed in TBS and then incubated for 30 min with the appropriate biotinylated (single-stain) or horseradish peroxidase (HRP; double-stain)-linked secondary antibody (DAKO, Ely, U.K.) diluted 1:500 in BSA/TBS. Following washes in TBS, single-stained sections were incubated with avidin-biotin-HRP complex (Vector Laboratories or DAKO) according to the manufacturer's instructions. Bound antibody, on both single- and double-stained sections, was visualized using 3,3-diaminobenzidine tetrahydrochloride (DAB; DAKO). Negative controls, omitting the primary antisera, were included in each experiment (Figs. 1 and 2, insets).
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Double-Staining Using Fast Blue
Following incubation with the second primary antibody, sections were washed in TBS and then incubated for 30 min with the appropriate biotinylated secondary antibody diluted 1:500 in BSA/TBS. Sections were again washed in TBS, then incubated with avidin-biotin-alkaline phosphatase complex (DAKO); bound antibody was visualized with Fast Blue (Sigma). Single (DAB)-stained sections were counterstained with hematoxylin, dehydrated, and mounted with Pertex (Cell Path, Hemel Hempstead, U.K.); double-stained sections were mounted in Permafluor (Beckman Coulter, High Wycombe, U.K.). Both were visualized by light microscopy.
Immunofluorescence
Immunofluorescence was used both for double-staining (CHK2/ MAGE-A4 and OCT4/MAGE-A4) and for triple-staining with antibodies against CHK2, MAGE-A4, and C-KIT. For the double- and triple-staining experiments, the antibodies were used at the following dilutions: anti-MAGE-A4, 1:30; anti-C-KIT, 1:100; anti-CHK2, 1:40; and anti-OCT4, 1: 1000. For double-staining with CHK2/MAGE-A4, the MAGE-A4 was visualized by Streptavidin-488 (Molecular Probes, Leiden, The Netherlands) via a biotinylated rabbit anti-mouse secondary antibody, whereas CHK2 was visualized by tyramide-enhanced Cy5 via an HRP-conjugated rabbit anti-mouse immunoglobulin G2
secondary antibody. For double-staining with OCT4/MAGE-A4, the MAGE-A4 was detected with Streptavidin Alexafluor 647 (Molecular Probes) via a biotinylated rabbit anti-mouse secondary antibody, and OCT4 was visualized by tyramide-enhanced Cy3 via an HRP-conjugated rabbit anti-goat secondary antibody.
To localize C-KIT, MAGE-A4, and CHK2 in the same section, the three primary antibodies and their respective detection systems were applied to the sections sequentially. First, C-KIT primary antibody was applied to the section and detected by a directly labeled goat anti-rabbit-Alexafluor546 secondary antibody (Molecular Probes). This was followed by MAGE-A4 primary antibody detected as above by Streptavidin-488, this time via a biotinylated goat anti-mouse secondary antibody. Finally, anti-CHK2 antibody was applied to the section and was detected as described above for the double-staining.
Image Capture
Nonfluorescent images were photographed using a Provis microscope (Olympus Optical, London, U.K.) and a Kodak DCS330 digital camera (Eastman Kodak, Rochester, NY). Fluorescent images were captured using a Zeiss LSM Axiovert 100M confocal microscope (Carl Zeiss Ltd., Welwyn Garden City, U.K.). Images were compiled using Photoshop 7 (Adobe, Mountain View, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The somatic components of human fetal testis were identified using specific antibodies, and the structural organization in the first- and second-trimester samples were compared (Fig. 1). In all samples obtained during the second trimester (14–19 wk), Sertoli cells that were immunopositive for mullerian-inhibiting substance (MIS) [25] were identified within clearly defined testicular cords (Fig. 1, b and c). The surrounding interstitial portion of the testis contained populations of functional Leydig cells (3-ß-hydroxysteroid dehydrogenase [3ßHSD]) (Fig. 1, e and f). Most cells that were immunopositive for androgen receptor (AR) were located around the base of the seminiferous cords (putative peritubular myoid cells) (Fig. 1, h and i). All samples from the first trimester (7–9 wk) were immunonegative for 3ßHSD and AR; the examples in Figure 1, d and g, are taken from the same fetus, the gestational age of which was estimated to be 64 days. In this testis (Fig. 1a), MIS-immunopositive cells are present, but testis cords are not as well defined as in samples from the second trimester. In the individual samples from the first trimester, the degree of cord formation based on the pattern of MIS staining was highly variable, ranging from none (54 days) to well defined (63 days). No staining was observed when the primary antibodies were replaced by normal rabbit serum (Fig. 1i, inset).
Differential Expression of Proteins by Germ Cells During the First and Second Trimesters
In first-trimester testes, expression of PLAP was detected in most germ cells (Fig. 2, day), whereas immunolocalization of CHK2 was variable between samples, with some being immunonegative. In those testes that were immunopositive for CHK2 (e.g., Fig. 2a), most of the germ cells had intense immunopositive nuclear staining. Cells that were immunopositive for C-KIT (Fig. 2g, arrow), MAGE-A4 (Fig. 2j), and PCTAIRE-1 (not shown) were rare, and no immunopositive cells were detected in some samples.
During the second trimester, the proportions of cells that were immunopositive for the same panel of antibodies was dependent on the gestational age of the fetus (Table 2). For example, at 14 wk of gestation (Fig. 2, b, e, h, and k), most germ cells were immunopositive for CHK2, PCTAIRE-1, or C-KIT, but few cells were immunopositive for PLAP or MAGE-A4. However, by 19 wk of gestation (Fig. 2, c, f, i, and l), although many CHK2-positive cells were still present, cells that were immunopositive for C-KIT or PCTAIRE-1 appeared to be reduced compared to 14 wk, and PLAP-immunopositive cells were rare. In contrast, a striking increase in the number of MAGE-A4-immunopositive cells was detected in testes collected on and after the 17th week of gestation; the immunopositive reaction was intense and usually associated with groups of germ cells at the periphery of the seminiferous cords. An example of this staining pattern can be seen in Fig. 2l.
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Morphological Appearance of Germ Cells Within Human Fetal Testis During the Second Trimester
In sections of testes from the first trimester, germ cells were easily distinguished from the Sertoli cells, because they had larger, rounder nuclei. They also all had a similar morphology, with a high nuclear:cytoplasmic ratio. In contrast, variation in the morphological appearance of the germ cells within the individual seminiferous cords was evident in sections from the second trimester (Fig. 3). Three populations of germ cells were identified based on the appearance of their nuclei and their nuclear:cytoplasmic ratio. Cells in population 1 had round nuclei containing a prominent nucleolus but very little cytoplasm (labeled 1 in Fig. 3). Those in population 2 often appeared in pairs at the periphery of the cords; although their nuclei contained a prominent nucleolus, they had a larger volume of cytoplasm than the cells in population 1. Cells in population 3 were larger than those in the other two populations. Because of the presence of more cytoplasm, their nuclei did not contain such a prominent nucleolus as populations 1 or 2, and these cells were typically found in groups at the periphery of the cord (labeled 3 in Fig. 3). Population 3 was particularly noticeable in sections taken from testes at 18 and 19 wk of gestation.
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Identification of Germ Cell Subpopulations by Immunohistochemical Profiling
OCT 4 OCT4 was immunolocalized to the nuclei of the majority of germ cells in the testes from the first trimester (Fig. 4, a and b). In testes from the second trimester (Fig. 4, c and d), OCT4 immunopositive staining was restricted to a subset of germ cell nuclei that, based on their morphological appearance, were identified as belonging to those of population 1.
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OCT 4/C-KIT Dual immunohistochemistry revealed that OCT4-immunopositive cells (Fig. 4, e and f, blue nuclei) expressed C-KIT on their surface. Germ cells that were OCT4neg/C-KITneg were identified in the same sections (Fig. 4f, arrows).
CHK2/C-KIT Three staining patterns were observed: CHK2pos/C-KITpos (Fig. 5, a and b, blue nuclei, brown membrane), CHK2pos/C-KITneg (Fig. 5b, inset), and CHK2neg/C-KITneg (Fig. 5a, arrows).
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MAGE-A4/C-KIT Costaining with antibodies against MAGE-A4 and C-KIT resulted in identification of separate groups of germ cells. Coexpression of the two proteins was not observed in either the first or second trimester (Fig. 5, c and d).
MAGE-A4/CHK2 CHK2 was detected in most germ cells (Fig. 6a), but coexpression of MAGE-A4 was only seen in a subset of these CHK2pos/MAGEpos) (Fig. 6a, arrows).
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OCT4/MAGE-A4 The OCT4- and MAGE-A4-immunopositive germ cell populations were distinct, and the two proteins were never coexpressed (Fig. 6, b and c).
CHK2/C-KIT/MAGE-A4 Triple-staining resulted in identification of three different types of CHK2 immunopositive cells: CHK2pos/C-KITpos (Fig. 6, day, e, asterisks), CHK2pos/MAGE-A4pos (Fig. 6, day, e, arrows), and CHK2pos/C-KITneg/MAGE-A4neg (Fig. 6e, blue nuclei with no membrane staining).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunolocalization of proteins expressed in the germ cell population revealed that although germ cells in the first trimester appeared to represent a largely homogeneous population, in the second trimester a number of distinct subpopulations were present. The CHK2-positive cells were identified in both first- and second-trimester samples; some first-trimester samples were immunonegative, suggesting that expression of this protein may be switched on at the time of cord formation. CHK2 is a serine/threonine kinase component of the S and G2/M replication checkpoint that may be involved in cell-cycle arrest in response to DNA damage [20]. CHK2 is also expressed in adult germ cells, but it is rapidly down-regulated as they enter meiosis [12]. In fetal life, expression of CHK2 in most germ cells may reflect an important role in the maintenance of the genomic integrity of the germ line. The majority of germ cells in the first-trimester testes appeared to be PLAP positive; however, in the second trimester, PLAP-positive cells were rare. The patterns of expression of C-KIT and PCTAIRE-1 were similar, with a reduction in the proportion of positive germ cells in 17- to 19-wk samples compared to less mature samples. Mice that lack a functional C-KIT receptor contain less germ cells because of reduced survival of migratory PGCs [18]; it is assumed that expression of C-KIT is also essential for PGC migration in humans, which would be consistent with detection of the protein on the surface of the OCT4-positive germ cells. Studies in rats have demonstrated that the kit ligand is a prosurvival factor for germ cells [31], and expression of C-KIT by human fetal germ cells may contribute to their survival. Detection of MAGE-A4 in fetal germ cells was associated with the appearance of groups of cells at the periphery of the cords.
The variable pattern of expression of putative germ cell markers in the tissues that we examined prompted us to determine whether this might be correlated with germ cell morphology. Data published 20–30 yr ago [2, 13–15], in which germ cell morphology was determined by electron microscopy, had described a variety of germ cell types. In the present study, consistent with these observations, we noted that germ cells with distinctive morphologies (small, round, and single vs. larger, peripheral groups) were present in second-trimester samples. OCT4 (POU5F1 [19]), a transcription factor that is expressed in pluripotent embryonic stem cells, was immunolocalized to the nuclei of most germ cells in first-trimester testes, consistent with reports that pluripotent embryonic germ (EG) cell lines can be derived by culturing PGCs (for review, see [32]). Notably, we found that expression of OCT4 persisted in a subpopulation of germ cells during the second trimester and that these cells also expressed C-KIT on their surface. Morphologically, this germ cell population was small and round, with a high nuclear:cytoplasmic ratio, and this fit the criteria proposed by Fukuda et al. [13] for classification as a gonocyte. Fluorescent colocalization studies revealed that the OCT4/C-KIT-positive cells were distinct from those that were MAGE-A4-positive. The morphology of the MAGE-A4-positive cells was strikingly similar to those classified as prespermatogonia by Fukuda et al., and we believe these represent a population of fetal germ cells that have differentiated from the pluripotent OCT4-positive gonocytes. In mice, expression of Oct4 in germ cells has been studied using a transgene in which expression of green fluorescent protein was driven by an 18-kilobase fragment encompassing the Oct-4 gene [9, 33]. In the fetal testes, OCT4-positive germ cells were mitotically active and expressed C-KIT at Embryonic Day 11.5. By Embryonic Day 16.5, mitotic activity had ceased, and C-KIT expression was down-regulated. On the day of birth, all the OCT4-positive germ cells were C-KIT-negative [34]. These results suggest that in contrast to those obtained during the present study, in mice, expression of C-KIT is not correlated with expression of OCT4.
A number of investigators have examined germ cell proliferation in the human testis. For example, Bendsen et al. [35] reported that between 6- and 9-wk postconception (8– 11 wk of gestation), the number of germ cells in the testis increased from approximately 3000 to 30 000, although the ratio of germ cells to somatic cells remained constant. In third-trimester samples, the number of germ cells has been estimated to be 3.8 x 106, which is consistent with further mitotic activity in the second trimester [36]. Wartenberg [2] classified the fetal germ cell population into M prospermatogonia (proliferating), T1 spermatogonia (nonproliferating), and T2 prospermatogonia (those T spermatogonia that had resumed mitotic activity). In follow-up studies, we have been examining the mitotic activity of human fetal germ cells using antibodies specific to proliferating cell nuclear antigen (PCNA) [37] and phosphorylated histone H3 [38]. These studies have revealed that the MAGE-A4-positive cell population (prespermatogonia) did not express PCNA, although staining was readily detectable in the nuclei of somatic cells and in the other germ cells. We did not identify germ cells equivalent to the T2 prospermatogonia in the second-trimester samples we examined.
It is now commonly accepted that testicular germ cell tumors of adolescents and young adults arise from a common precursor cell type known as carcinoma in situ (CIS) [39, 40]. Observations such as the high level of expression of C-KIT and PLAP in CIS cells [17, 41] have supported evidence that these cells share morphological and functional characteristics with fetal germ cells. A comparison between the patterns of protein expression seen in the germ cell subpopulations identified in the current study and the patterns of protein expression reported for CIS cells appears to provide further data relevant to determining the cells that contribute to the CIS population. In a series of studies, CIS cells have been shown to be immunopositive for CHK2 [12], DAZL [42], PLAP [43–45], C-KIT [41], and VASA [46]. A recent study also demonstrated that CIS cells immunopositive for C-KIT also express OCT4 [47]. The authors suggested that the expression of OCT4 in the CIS population is consistent with the maintenance of pluripotent potential in this cell type. The results obtained in the present study have demonstrated that the gonocyte population of fetal germ cells (population 1) (Table 3) are OCT4pos/C-KITpos/PLAPpos/CHK2pos, and this would be consistent with the idea that the CIS cell is the malignant counterpart of this germ cell population (for review, see [48]).
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The MAGE-A4 has been detected in CIS cells and germ cell tumors [49]; those authors reported that although MAGE-A4 could be detected in most tissue sections, only half the cells identified as CIS based on their morphology were MAGE-A4-positive. In our studies on the fetal testes, we have never found a germ cell that coexpressed C-KIT and MAGE-A4, and our preliminary observations (unpublished) suggest that although C-KITpos and MAGE-A4pos CIS cells can be detected, the two proteins are not coexpressed in the same cell. One interpretation of these findings is that cells with a CIS phenotype can develop not only from gonocytes but also from the OCT4neg/C-KITneg/ MAGE-A4pos population (prespermatogonia). Alternatively, it is possible that some CIS cells undergo partial differentiation that is characterized by loss of expression of OCT4 and onset of expression of MAGE-A4.
In conclusion, our findings extend those of Fukuda et al. [13], who used morphological criteria to identify three populations of human germ cells in testes obtained during the second trimester. In agreement with their findings, we propose that the human germ cells should be classified as gonocytes, intermediate cells, and prespermatogonia, depending on their appearance and the expression of three of the proteins evaluated by immunohistochemistry. The characteristics of the cell types are outlined in Table 3. In summary, gonocytes are a population of single, round cells characterized as OCT4pos/C-KITpos/MAGE-A4neg. Intermediate cells usually occur in pairs and are characterized as C-KITneg/MAGE-A4neg/OCT4low/neg. Both these cell populations are PCNA-positive and, therefore, would be grouped together as M prospermatogonia according to the criteria outlined by Wartenberg [2]. A third population of "prespermatogonia" usually occurs as groups of cells at the periphery of the cords and is characterized as OCT4neg/ C-KITneg/MAGE-A4pos; these cells are PCNA-negative and, in gross appearance, appear similar to the cells classified as T1 spermatogonia by Wartenberg [2]. In the testes recovered during the first trimester, the majority of germ cells were classified as gonocytes; however, during the second trimester, distinct populations of gonocytes, intermediate cells, and prespermatogonia were detected within the same cord. The proportion of prespermatogonia increased by the end of the second trimester, which is consistent with functional differentiation of germ cells during this period of pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 11 February 2004.
First decision: 1 March 2004.
Accepted: 3 August 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxylase, C17,20-lyase (P450c17), and the receptor for luteinizing hormone (LH) in the fetal rat testis suggests that the onset of Leydig cell steroid production is independent of LH action. Biol Reprod 1998 58:520-525This article has been cited by other articles:
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  R.T. Mitchell, G. Cowan, K.D. Morris, R.A. Anderson, H.M. Fraser, K.J. Mckenzie, W.H.B. Wallace, C.J.H. Kelnar, P.T.K. Saunders, and R.M. Sharpe Germ cell differentiation in the marmoset (Callithrix jacchus) during fetal and neonatal life closely parallels that in the human Hum. Reprod., August 11, 2008; (2008) den295v1. [Abstract] [Full Text] [PDF]
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 P. A. Fowler, S. Cassie, S. M. Rhind, M. J. Brewer, J. M. Collinson, R. G. Lea, P. J. Baker, S. Bhattacharya, and P. J. O'Shaughnessy Maternal Smoking during Pregnancy Specifically Reduces Human Fetal Desert Hedgehog Gene Expression during Testis Development J. Clin. Endocrinol. Metab., February 1, 2008; 93(2): 619 - 626. [Abstract] [Full Text] [PDF]
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R. Lambrot, H. Coffigny, C. Pairault, A.-C. Donnadieu, R. Frydman, R. Habert, and V. Rouiller-Fabre Use of Organ Culture to Study the Human Fetal Testis Development: Effect of Retinoic Acid J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2696 - 2703. [Abstract] [Full Text] [PDF]
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P. A. Kumar, N. Pitteloud, P. A.M. Andrews, A. Dwyer, F. Hayes, W. F. Crowley Jr, and M. Dym Testis morphology in patients with idiopathic hypogonadotropic hypogonadism Hum. Reprod., April 1, 2006; 21(4): 1033 - 1040. [Abstract] [Full Text] [PDF]
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HSD-positive Leydig cells (Lc; e and f) and AR-immunopositive peritubular cell populations were detected. The morphological appearance of first-trimester samples was variable, and although all contained MIS-positive immunostaining (a, arrows), clearly defined testis cords were rare. Immunopositive staining for 3ßHSD and AR was not detected in the interstitium (In; b and c). The inset in i shows a typical control in which normal rabbit serum was used in place of primary antibody. Magnification, x40
