Héctor E. Chemes

Ontogeny of the androgen receptor expression in the fetal and postnatal testis: its relevance on Sertoli cell maturation and the onset of adult spermatogenesis.

From fetal life to adulthood, the testis evolves through maturational phases showing specific morphologic and functional features in its different compartments. The seminiferous cords contain Sertoli and germ cells, surrounded by peritubular cells, and the interstitial tissue contains Leydig cells and connective tissue. Sertoli cells secrete anti‐Müllerian hormone (AMH), whereas Leydig cells secrete androgens. In the fetal and early postnatal testis, Leydig cells actively secrete androgens. Sertoli cells are morphologically and functionally immature—e.g., they secrete high levels of AMH—and germ cells proliferate by mitosis but do not enter meiosis. During infancy and childhood, Leydig cells regress and testosterone secretion declines dramatically. Sertoli cells remain immature and spermatogenesis is arrested at the premeiotic stage. At puberty, Leydig cells differentiate again, and testosterone concentration increases and provokes Sertoli cell maturation—e.g., down‐regulation of AMH expression—and germ cells undergo meiosis, the hallmark of adult spermatogenesis driving to sperm production. An intriguing feature of testicular development is that, although testosterone production is as active in the fetal and early postnatal periods as in puberty, Sertoli cells and spermatogenesis remain immature until pubertal onset. Here, we review the ontogeny of the androgen receptor expression in the testis and its impact on Sertoli cell maturation and the onset of pubertal spermatogenesis. We show that the absence of androgen receptor expression in Sertoli cells underlies a physiological stage of androgen insensitivity within the male gonad in the fetal and early postnatal periods. Microsc. Res. Tech., 2009.

Physiological Androgen Insensitivity of the Fetal, Neonatal, and Early Infantile Testis Is Explained by the Ontogeny of the Androgen Receptor Expression in Sertoli Cells

CONTEXT Although gonadotropins and testosterone are high in the fetal/early postnatal periods, Sertoli cells remain immature and spermatogenesis does not progress. We hypothesized that Sertoli cells do not respond to testosterone because they do not express the androgen receptor. OBJECTIVE The objective of the study was to describe the precise ontogeny of androgen receptor expression in the human testis from fetal life through adulthood. DESIGN This was an immunohistochemical study on testicular biopsies from fetal, neonatal, prepubertal, pubertal, and adult human testes. MAIN OUTCOME MEASURES Quantification of androgen receptor expression in Sertoli cells was measured. Evaluation of androgen receptor expression in peritubular and interstitial cells as well as anti-Müllerian hormone and inhibin-alpha was also performed. RESULTS Androgen receptor expression was first observed in the nuclei of few Sertoli cells at the age of 5 months. Labeling was weak in 2-15% of Sertoli cells until 4 yr of age and progressively increased thereafter. High levels of androgen receptor expression were observed in more than 90% from the age of 8 yr through adulthood. Androgen receptor was positive in peritubular cells and variable in interstitial cells. Anti-Müllerian hormone immunolabeling was strong in all Sertoli cells from fetal life throughout prepuberty and weakened progressively as spermatogenesis developed. Inhibin-alpha expression was detected in all Sertoli cells from fetal life through adulthood. CONCLUSIONS A lack of androgen receptor expression could explain a physiological Sertoli cell androgen insensitivity during fetal and early postnatal life, which may serve to protect the testis from precocious Sertoli cell maturation, resulting in proliferation arrest and spermatogenic development.

Regulation of follicular luteinization by a gonadotropin-releasing hormone agonist: Relationship between steroidogenesis and apoptosis

The purpose of this study was to evaluate the effects of GnRH‐analog (Leuprolide acetate, LA) administration on follicular luteinization in equine chorionic gonadotropin plus human chorionic gonadotropin (eCG + hCG)‐superovulated prepubertal treated rats. Results indicate that LA treatment decreases circulating levels of progesterone (P) and P accumulation in collagenase‐dispersed ovarian cell cultures, though estradiol(E2) production is increased. These data suggest that cells from the LA group may be less luteinized following gonadotropin treatment. Studies performed on histological ovarian sections after different times of eCG administration showed that LA injections produce lower amounts of corpora lutea and antral follicles, and a greater number of atretic and preantral follicles. The basal and LH‐stimulated P and progestagen accumulations are decreased in incubations of corpora lutea isolated from the LA group. In addition, the mitochondrial cholesterol side‐chain cleavage (P450SCC) levels in corpora lutea from LA‐treated rats are reduced, indicating that the decrease in P production observed is due in part to an alteration in the steroidogenic luteal capability. Immunocytochemical localization of nuclei exhibiting DNA fragmentation by the technique of terminal deoxynucleotidyl transferase end‐labeling showed that LA treatment causes an increase in the number of apoptotic cells in preantral and antral follicles at all times studied (1, 2, 4, or 7 days of LA administration). A similar effect, though less pronounced, was observed in corpora lutea. It is concluded that LA treatment produces a failure in the steroidogenic luteal capability and an increase of apoptotic mechanisms in the ovary, producing as a consequence an interference in the follicular recruitment, growth, and luteinization induced by gonadotropins. Mol. Reprod. Dev. 51:287–294, 1998.

Tales of the Tail and Sperm Head AchesChanging concepts on the prognostic significance of sperm pathologies affecting the head, neck and tail

This article presents an update on the variable prognostic significance of different sperm pathologies in patients with severe male factor infertility due to morphology and motility disorders. Severe asthenozoospermia is one of the leading causes of male infertility as spermatozoa cannot reach the oocyte and/or penetrate normally. Identifying structural causes of sperm immotility was of great concern before the advent of intracytoplasmic sperm injection (ICSI), because immotility was the limiting factor in the treatment of these patients. In these cases, in vitro methods are used to identify live spermatozoa or stimulate sperm motility to avoid selection of non-viable cells. With these advances, fertilization and pregnancy results have improved dramatically. The identification of genetic phenotypes in asthenozoospermia is important to adequately inform patients of treatment outcomes and risks. The one sperm characteristic that seriously affects fertility prognosis is teratozoospermia, primarily sperm head and neck anomalies. Defects of chromatin condensation and acrosomal hypoplasia are the two most common abnormalities in severe teratozoospermia. The introduction of microscopic methods to select spermatozoa and the development of new ones to evaluate sperm quality before ICSI will assure that ultrastructural identification of sperm pathologies will not only be of academic interest, but will also be an essential tool to inform treatment choice. Herein, we review the differential roles played by sperm components in normal fertilization and early embryo development and explore how assisted reproductive technologies have modified our concepts on the prognostic significance of sperm pathologies affecting the head, neck, mid-piece and tail.

Early manifestations of testicular dysgenesis in children: pathological phenotypes, karyotype correlations and precursor stages of tumour development.

Testicular dysgenesis derives from abnormal gonadal development caused by chromosome aberrations/mosaicisms or mutations/deletions in SRY or other genes responsible for testicular differentiation. Dysgenetic male pseudohaermaphroditism has bilateral dysgenetic testes characterized by a cortical network of anastomosing seminiferous cords that penetrate a thin albuginea. In asymmetric gonadal differentiation (or Mixed Gonadal Dysgenesis) a dysgenetic testis associates with a streak gonad with primitive sex cords embedded in an ovarian‐like stroma. Uni‐ or bilateral ovotestes identify true haermaphroditism. Fluorescent in situ hybridisation studies demonstrate that the sex chromosomes of mosaic patients do not distribute homogeneously in asymmetric gonads. 45,X lines predominate over 46,XY in streak gonads, while the relationship between these two is more equivalent in dysgenetic testes, suggesting that testicular or streak differentiation is related to the balance between X0 and XY lines. Testicular dysgenesis is more severe when there is a frank predominance of X0 or XX cells. Higher percentages of XY cells coincide with lesser degrees of dysgenesis. DNA densitometry indicate a higher incidence of neoplastic transformation than previously anticipated. Various specimens showed clear aneuploid histograms but no clear indication of a cytological CIS phenotype. There was a wide cytological variation in aneuploid germ cells, ranging from normally looking big infantile spermatogonia to gonocyte/CIS cells. Aneuploidy probably precedes the full expression of the CIS phenotype. In case of doubt we recommend DNA densitometry to either confirm or discard their neoplastic nature. The earliest recognizable change in germ cell tumorigenesis is probably the polyploidisation of fetal germ cells, followed by the expression of the CIS phenotype in isolated germ cells scattered along infantile seminiferous tubules that later proliferate to give an adult type CIS pattern.

The making of abnormal spermatozoa: cellular and molecular mechanisms underlying pathological spermiogenesis

Fertilization in mammals occurs via a series of well-defined events in the secluded environment of the female reproductive tract. The mode of selection of the fertilizing spermatozoon nevertheless remains unknown. As has become evident during in vitro fertilization by sperm microinjection into the oocyte, abnormal spermatozoa can successfully fertilize oocytes. Under these extreme conditions, post-fertilization events, early embryonic development and implantation are significantly compromised indicating that the contribution of spermatozoa extends beyond sperm penetration. Microscopic identification of normal spermatozoa is a well-standardized procedure but insights into the mechanisms that lead to aberrant sperm differentiation and into the subcellular nature of sperm abnormalities have only recently begun to be obtained. The spermatozoon is the result of a complex development in which spermatid organelles give rise to various structural components with characteristic functions. Similar to other differentiated cells, the spermatozoon has a specific pathology that is most clearly identified by ultrastructural evaluation coupled with immunocytochemistry and molecular techniques. This multidisciplinary approach allows the precise characterization of sperm abnormalities, including structural, molecular and functional aspects. We summarize here studies of the physiopathology of spermiogenesis in two abnormal sperm phenotypes of infertile men: dysplasia of the fibrous sheath and acephalic spermatozoa/abnormal head-tail attachment. The characterization of the abnormalities of the tail cytoskeleton and centrioles has uncovered aspects of the subcellular basis of pathological spermiogenesis, has suggested experimental approaches to explore the nature of these anomalies and has opened the way for genetic studies that will ultimately lead to the design of the therapeutic tools of the future.

Sertoli cell proliferations of the infantile testis: an intratubular form of Sertoli cell tumor?

We report on six boys with intratubular Sertoli cell proliferations (ISCPs), studied by routine histologic methods, electron microscopy, and immunohistochemistry of anti-müllerian hormone (AMH), inhibin &agr;-subunit, 3&bgr;-hydroxysteroid dehydrogenase (3&bgr;-HSD), proliferative cellular nuclear antigen, and p53, and carefully followed for extended periods with periodic clinical examinations, testicular ultrasonographies, and determinations of serum levels of AMH and inhibin B. Peutz–Jeghers syndrome was found in four of six patients, and gynecomastia occurred in five of six patients. One boy had isosexual pseudoprecocity. ISCPs were observed as multiple foci of seminiferous tubules with large and proliferated Sertoli cells replacing germ cells and limited by the basement membrane. Mitotic figures, atypia, and/or interstitial invasion were not observed. Bilateral ISCPs were the only pathologic finding in three patients (patient nos. 1–3) and were associated with a microscopic tumor that resembled a large-cell calcifying Sertoli cell tumor (LCCSCT) in a fourth patient (patient no. 4). In the two remaining patients (patient nos. 5 and 6) ISCPs and LCCSCT were found in both testes. Ultrastructural examination showed large Sertoli cells, with round nuclei, sparse organelles, and some glycogen. Inhibin &agr;-subunit immunolocalization was positive in the five patients in whom it was determined (patient nos. 2–6), AMH was positive in those ISCPs associated with tumors (patient nos. 4–6) and negative in isolated ISCPs (patient nos. 2 and 3); 3&bgr;-HSD and PCNA were variable, and p53 was negative in all ISCPs. Patient nos. 1–4 have been followed for 2–19 years. One of them is currently entering puberty, the other two have already completed puberty and have testes of normal size, and the remaining one is an adult with clinically normal testes and sperm production. None of these patients had evidence of tumor development during follow-up as shown by serial ultrasonographies and serum levels of AMH and inhibin B. Patient nos. 5 and 6 who had bilateral ISCPs and LCCSCT were orchidectomized and evolved for 2–10 years after surgery without tumor recurrence. The prognostic significance of ISCPs, particularly when they are the only pathologic finding in a testicular biopsy, is a matter of controversy. Based on the long normal evolution, we recommend a conservative approach to therapy. The bilateral and multicentric character of ISCPs and their association with Sertoli tumors and Peutz–Jeghers syndrome suggest that they represent either proliferative lesions with tumorigenic potential or the intraepithelial stage in the evolution of some testicular Sertoli cell tumors.

Acrosomal biogenesis in human globozoospermia: immunocytochemical, ultrastructural and proteomic studies

BACKGROUND Acrosome biogenesis is a key event in sperm differentiation that depends on the proper interaction between the Golgi complex and the nuclear envelope of early spermatids. We studied the development, structure and biochemical characteristics of human acrosomes in germ cells and spermatozoa from testicular biopsies and semen samples of fertile men and patients with acrosomeless spermatozoa (globozoospermia). A set of proteins collectively known as the perinuclear theca (PT), which has been related to acrosomal development in many mammalian species, were also investigated. METHODS We evaluated spermatozoa from five males with globozoospermia and six fertile men, and immature germ cells from testicular biopsies of one globozoospermic patient and three men with obstructive azoospermia. Samples were assessed by transmission electron microscopy, immunofluorescence microscopy, ultrastructural immunocytochemistry and proteomic analysis by western blot. RESULTS In normal spermiogenesis, the development of the acrosome depends on the correct formation of Golgi-derived proacrosomal vesicles and simultaneous modifications in the nuclear envelope. PT proteins are consistently found in proacrosomic vesicles, localize underneath the acrosome and expand over the nuclear surface along acrosome biogenesis. In fertile men, the PT is composed of six proteins, similar to those previously described for other mammals (16, 22, 29, 34, 50 and 68 kDa). In globozoospermia, abnormal proacrosomal vesicles and paranuclear multivesicular and multilamellar structures were observed that resulted in acrosomes insufficiently developed or detached from the nuclear envelope. PT proteins, dissociated from the acrosomes, were ectopically localized in the cytoplasm. Proteomic analysis showed a significant decrease in all six PT proteins. CONCLUSIONS The alterations observed during early acrosome biogenesis in globozoospermia are due to anomalous development of Golgi-derived proacrosomic vesicles, failure of PT proteins to properly associate with the nuclear surface and significant deficiencies in specific PT components that are necessary for proper acrosome formation, implantation and expansion over the spermatid nucleus.

Morphometric study of the testicular interstitial tissue of the monkey Cebus apella during postnatal development

The purpose of this study was to evaluate the developmental changes of the Leydig cells and their precursors during postnatal development in the monkey Cebus apella. Four groups of monkeys were studied: neonatal, infantile, early pubertal and late pubertal. Light microscopy, immunocytochemistry, electron microscopy and stereological studies were performed to determine cytologic and cytochemical characteristics, volume density, absolute volume and cell counts of Leydig cells. In the interstitial tissue two components were recognized: specific interstitium comprising mature and immature Leydig cells and differentiating Leydig cell precursors, and non-specific interstitium including connective tissue and blood vessels. Mature Leydig cells were polygonal with a round, euchromatic nucleus and abundant cytoplasm. Immature Leydig cells were more elongated and the nucleus showed more heterochromatin. Mature and immature Leydig cells showed either a pale- or a dark-stained cytoplasm. Pale Leydig cells showed abundant smooth endoplasmic reticulum (SER), mitochondria with tubular cristae and glycogen granules. The SER of dark Leydig cells consisted of abundant flat cisternae, only few glycogen inclusions and abundant lipid droplets. All Leydig cells were intensely reactive for 3beta-hydrohysteroid dehydrogenase (3beta-HSD). Some peritubular cells acquired nuclear and cytoplasmic characteristics that indicated that they were differentiating to Leydig cells, as evidenced by the strong 3beta-HSD positivity found in scattered elongated cells of the peritubular tissue. Absolute interstitial volume increased from birth to the end of puberty due to an increment in Leydig cells numbers and size. The mature and immature Leydig cell populations showed a different evolution during postnatal development. While immature Leydig cells increased 7-fold from the neonatal to the early pubertal period and increased at a lower rate during puberty, mature Leydig cells remained stable until early puberty and increased significantly during late pubertal development.

The axoneme: the propulsive engine of spermatozoa and cilia and associated ciliopathies leading to infertility.

This review article provides a critical analysis of the structure and molecular mechanisms of the microtubule axoneme of cilia and sperm flagella and their associated elements required for male fertility.A broad range of genetic and molecular defects (ciliopathies) are considered in the context of human diseases involving impaired motility in cilia and sperm flagella, providing provocative thought for future research in the area of male infertility.