STUDY QUESTION: What are the causative genetic variants in patients with male infertility due to severe sperm motility disorders? SUMMARY ANSWER: We identified high confidence disease-causing variants in multiple genes previously associated with severe sperm motility disorders in 10 out of 21 patients (48%) and variants in novel candidate genes in seven additional patients (33%). WHAT IS KNOWN ALREADY: Severe sperm motility disorders are a form of male infertility characterised by immotile sperm often in combination with a spectrum of structural abnormalities of the sperm flagellum that do not affect viability. Currently, depending on the clinical sub-categorisation, up to 50% of causality in patients with severe sperm motility disorders can be explained by pathogenic variants in at least 22 genes. STUDY DESIGN, SIZE, DURATION: We performed exome sequencing in 21 patients with severe sperm motility disorders from two different clinics. PARTICIPANTS/MATERIALS, SETTING, METHOD: Two groups of infertile men, one from Argentina (n = 9) and one from Australia (n = 12), with clinically defined severe sperm motility disorders (motility <5%) and normal morphology values of 0-4%, were included. All patients in the Argentine cohort were diagnosed with DFS-MMAF, based on light and transmission electron microscopy. Sperm ultrastructural information was not available for the Australian cohort. Exome sequencing was performed in all 21 patients and variants with an allele frequency of <1% in the gnomAD population were prioritised and interpreted. MAIN RESULTS AND ROLE OF CHANCE: In 10 of 21 patients (48%), we identified pathogenic variants in known sperm assembly genes: CFAP43 (3 patients); CFAP44 (2 patients), CFAP58 (1 patient), QRICH2 (2 patients), DNAH1 (1 patient) and DNAH6 (1 patient). The diagnostic rate did not differ markedly between the Argentinian and the Australian cohort (55% and 42%, respectively). Furthermore, we identified patients with variants in the novel human candidate sperm motility genes: DNAH12, DRC1, MDC1, PACRG, SSPL2C and TPTE2. One patient presented with variants in four candidate genes and it remains unclear which variants were responsible for the severe sperm motility defect in this patient. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: In this study, we described patients with either a homozygous or two heterozygous candidate pathogenic variants in genes linked to sperm motility disorders. Due to unavailability of parental DNA, we have not assessed the frequency of de novo or maternally inherited dominant variants and could not determine the parental origin of the mutations to establish in all cases that the mutations are present on both alleles. WIDER IMPLICATIONS OF THE FINDINGS: Our results confirm the likely causal role of variants in six known genes for sperm motility and we demonstrate that exome sequencing is an effective method to diagnose patients with severe sperm motility disorders (10/21 diagnosed; 48%). Furthermore, our analysis revealed six novel candidate genes for severe sperm motility disorders. Genome-wide sequencing of additional patient cohorts and re-analysis of exome data of currently unsolved cases may reveal additional variants in these novel candidate genes.

Abstract STUDY QUESTION Can exome sequencing identify new genetic causes of globozoospermia? SUMMARY ANSWER Exome sequencing in 15 cases of unexplained globozoospermia revealed deleterious mutations in seven new genes, of which two have been validated as causing globozoospermia when knocked out in mouse models. WHAT IS KNOWN ALREADY Globozoospermia is a rare form of male infertility characterised by round-headed sperm and malformation of the acrosome. Although pathogenic variants in DPY19L2 and SPATA16 are known causes of globozoospermia and explain up to 70% of all cases, genetic causality remains unexplained in the remaining patients. STUDY DESIGN, SIZE, DURATION After pre-screening 16 men for mutations in known globozoospermia genes DPY19L2 and SPATA16, exome sequencing was performed in 15 males with globozoospermia or acrosomal hypoplasia of unknown aetiology. PARTICIPANTS/MATERIALS, SETTING, METHOD Targeted next-generation sequencing and Sanger sequencing was performed for all 16 patients to screen for single-nucleotide variants and copy number variations in DPY19L2 and SPATA16. After exclusion of one patient with DPY19L2 mutations, we performed exome sequencing for the 15 remaining subjects. We prioritised recessive and X-linked protein-altering variants with an allele frequency of <0.5% in the population database GnomAD in genes with an enhanced expression in the testis. All identified candidate variants were confirmed in patients and, where possible, in family members using Sanger sequencing. Ultrastructural examination of semen from one of the patients allowed for a precise phenotypic characterisation of abnormal spermatozoa. MAIN RESULTS AND ROLE OF CHANCE After prioritisation and validation, we identified possibly causative variants in eight of 15 patients investigated by exome sequencing. The analysis revealed homozygous nonsense mutations in ZPBP and CCDC62 in two unrelated patients, as well as rare missense mutations in C2CD6 (also known as ALS2CR11), CCIN, C7orf61 and DHNA17 and a frameshift mutation in GGN in six other patients. All variants identified through exome sequencing, except for the variants in DNAH17, were located in a region of homozygosity. Familial segregation of the nonsense variant in ZPBP revealed two fertile brothers and the patient’s mother to be heterozygous carriers. Paternal DNA was unavailable. Immunohistochemistry confirmed that ZPBP localises to the acrosome in human spermatozoa. Ultrastructural analysis of spermatozoa in the patient with the C7orf61 mutation revealed a mixture of round heads with no acrosomes (globozoospermia) and ovoid or irregular heads with small acrosomes frequently detached from the sperm head (acrosomal hypoplasia). LIMITATIONS, REASONS FOR CAUTION Stringent filtering criteria were used in the exome data analysis which could result in possible pathogenic variants remaining undetected. Additionally, functional follow-up is needed for several candidate genes to confirm the impact of these mutations on normal spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS Our study revealed an important role for mutations in ZPBP and CCDC62 in human globozoospermia as well as five new candidate genes. These findings provide a more comprehensive understanding of the genetics of male infertility and bring us closer to a complete molecular diagnosis for globozoospermia patients which would help to predict the success of reproductive treatments. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by The Netherlands Organisation for Scientific Research (918–15-667); National Health and Medical Research Council of Australia (APP1120356) and the National Council for Scientific Research (CONICET), Argentina, PIP grant 11220120100279CO. The authors have nothing to disclose.

Reduced serum testosterone (T), or hypogonadism, affects millions of men and is associated with many pathologies, including infertility, cardiovascular diseases, metabolic syndrome, and decreased libido and sexual function. Administering T-replacement therapy (TRT) reverses many of the symptoms associated with low T levels. However, TRT is linked to side effects such as infertility and increased risk of prostate cancer and cardiovascular diseases. Thus, there is a need to obtain T-producing cells that could be used to treat hypogonadism via transplantation and reestablishment of T-producing cell lineages in the body. T is synthesized by Leydig cells (LCs), proposed to derive from mesenchymal cells of mesonephric origin. Although mesenchymal cells have been successfully induced into LCs, the limited source and possible trauma to donors hinders their application to clinical therapies. Alternatively, human induced pluripotent stem cells (hiPSCs), which are expandable in culture and have the potential to differentiate into all somatic cell types, have become the emerging source of autologous cell therapies. We have successfully induced the differentiation of hiPSCs into either human Leydig-like (hLLCs) or adrenal-like cells (hALCs) using chemically defined culture conditions. Factors critical for the development of LCs were added to both culture systems. hLLCs expressed all steroidogenic genes and proteins important for T biosynthesis, synthesized T rather than cortisol, secreted steroid hormones in response to dibutyryl-cAMP and 22(R)-hydroxycholesterol, and displayed ultrastructural features resembling LCs. By contrast, hALCs synthesized cortisol rather than T. The success in generating hiPSC-derived hLLCs with broad human LC (hLC) features supports the potential for hiPSC-based hLC regeneration.

The present paper reviews in detail ultrastructural and molecular studies addressed to characterize different phenotypes of sperm pathology in sterile men. In each case ultrastructural, immunocytochemical, molecular and genetic information is provided to differentiate two main kinds of sperm pathologies: systematic phenotypes with known or suspected genetic origin and non-systematic ones, usually secondary to different pathologies of the male reproductive system. Special attention is paid to detailed ultrastructural features profusely illustrated with electron micrographs. Diagnostic and fertility prognostic values of these phenotypes are also discussed and, when possible, comparison with similar pathologies in mammals and birds are discussed.

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.

SummaryAll malignant testicular germ cell tumors (TGCT) of adult men are preceded by an in situ stage (CIS) of protracted evolution. The adult CIS is well characterized, but there is debate on the phenotype of infantile CIS, its distinction from delayed maturation of germ cells and prognostic potential. A large series of 43 patients with Disorders of Sex Development (DSD) and dysgenetic testes (90% ranging from neonates to 12 years, mean age 4.7 years), was studied by quantifying dysgenetic features, degree of germ cell abnormalities/atypia (GCA), expression of OCT 3/4 (a pluripotency‐undifferentiation marker), germ cell ploidy and evolution to CIS and invasive TGCT. Findings were compared with those of normal testes. The type of gonads present defined three groups of patients: bilateral testes (BT‐DSD, n = 21), one testis and one streak gonad (CT‐DSD, C for combined, n = 13), and ovarian‐testicular combinations (OT‐DSD, n = 9). There were 5 boys with infantile CIS, bilateral in 3 (total of 8 infantile CIS) and two patients with adult CIS, bilateral in one (total of 3 adult CIS). Two patients had bilateral seminomas one at 12–17 and the other at 23 years. Histological dysgenesis was significantly higher in CT‐DSD (p < 0.05), that had only 1 CIS. The highest frequency of GCA was in BT‐DSD (p < 0.05), which coincided with a total of 11CIS + Seminomas. In all patients, aneuploidy was significantly higher (63%) than diploidy (p < 0.02), and GCA were more frequent in aneuploid than in diploid samples (p < 0.02). All CIS and TGCT were OCT 3/4 positive. Finally, there was a significant association between the triad Aneuploidy + GCA + OCT 3/4 positivity and the incidence of CIS (Fisher Exact test p < 0.002, relative risk 7.0). The degree of testicular dysgenesis (derived from abnormal organization of Sertoli cells in fetal testicular cords) is inversely related to the incidence of CIS. Our data demonstrate that the combined use of OCT 3/4 expression, quantification of germ cell abnormalities‐atypia and ploidy in dysgenetic testes can satisfactorily identify infantile CIS with high risk of malignant evolution and set it aside from delayed germ cell maturation with lower or nil neoplastic potential.

A microscopia eletrônica de espermatozoides é uma ferramenta complementar da análise seminal que pode contribuir na interpretação clínica da astenozoospermia grave e da teratozoospermia e na investigação de infertilidade idiopática. Reportamos um caso de paciente com varicocele, submetido à varicocelectomia, com análise seminal ultraestrutural por microscopia eletrônica.

CONTEXT:Pheochromocytomas and paragangliomas (pheo/pgl) are neuroendocrine tumours derived from chromaffin cells. Although mostly benign, up to 26% of pheo/pgl will undergo malignant transformation. Reliable histological signs to differentiate benign pheo/pgl from malignant tumours are currently lacking. Increased IGF-1R expression has been shown during progression to metastatic phenotypes of several types of cancer.OBJECTIVE:To analyse the distribution and expression of the IGF-1R in pheo/pgl of different genetic origin and degree of malignancy.MEASUREMENTS:We studied the expression of the IGF-1R protein by immunohistochemistry, in 40 primary tumours from patients with pheo/pgl from different genetic aetiology (11 of 29 metastatic/nonmetastatic diseases).RESULTS:We found a strong association between increased expression of IGF-1R and malignant behaviour regardless of the age at diagnosis and the genetic aetiology. IGF-1R labelling was mostly weak in primary tumours from patients with nonmetastatic pheo/pgl. Conversely, intense IGF-1R labelling was predominant in cases of pheo/pgl with confirmed metastatic disease. The risk of metastases was 11·7 times higher if tumour IGF-1R labelling was intense independently of age at diagnosis. The probability of remaining free of metastases was higher in patients with pheo/pgl scored weak for IGF-1R at 60 months and more than twofold higher at 120 months of follow-up than in patients with intense IGF-1R labelling in their primary tumours.CONCLUSIONS:Our results strongly suggest that IGF-1R is associated with malignancy in familial pheo/pgl and that IGF-1R expression in the primary tumour might be a useful tool to detect those patients harbouring pheo/pgl who have an increased risk of metastasis.

Transmission electron microscopy (TEM) studies have provided the basis for an in-depth understanding of the cell biology and normal functioning of the testis and male gametes and have opened the way to characterize the functional role played by specific organelles in spermatogenesis and sperm function. The development of the scanning electron microscope (SEM) extended these boundaries to the recognition of cell and organ surface features and the architectural array of cells and tissues. The merging of immunocytochemical and histochemical approaches with electron microscopy has completed a series of technical improvements that integrate structural and functional features to provide a broad understanding of cell biology in health and disease. With these advances the detailed study of the intricate structural and molecular organization as well as the chemical composition of cellular organelles is now possible. Immunocytochemistry is used to identify proteins or other components and localize them in specific cells or organelles with high specificity and sensitivity, and histochemistry can be used to understand their function (i.e., enzyme activity). When these techniques are used in conjunction with electron microscopy their resolving power is further increased to subcellular levels. In the present chapter we will describe in detail various ultrastructural techniques that are now available for basic or translational research in reproductive biology and reproductive medicine. These include TEM, ultrastructural immunocytochemistry, ultrastructural histochemistry, and SEM.

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.

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.

IGFs are involved in malignant transformation and growth of several tissues, including the adrenal medulla. The present study was designed to evaluate the impact of IGF-I on pheochromocytoma development. We used a murine pheochromocytoma (MPC) cell line (MPC4/30) and an animal model with a reduction of 75% in circulating IGF-I levels [liver-IGF-I-deficient (LID) mice] to perform studies in vitro and in vivo. We found that, in culture, IGF-I stimulation increases proliferation, migration, and anchorage-independent growth, whereas it inhibits apoptosis of MPC cells. When injected to control and to LID mice, MPC cells grow and form tumors with features of pheochromocytoma. Six weeks after cell inoculation, all control mice developed sc tumors. In contrast, in 73% of LID mice, tumor development was delayed to 7-12 wk, and the remaining 27% did not develop tumors up to 12 wk after inoculation. LID mice harboring MPC cells and treated with recombinant human IGF-I (LID+) developed tumors as controls. Tumors developed in control, LID, and LID+ mice had similar histology and were similarly positive for IGF-I receptor expression. The apoptotic index was higher in tumors from LID mice compared with those from control mice, whereas vascular density was decreased. In summary, our work demonstrates that IGF-I has a critical role in maintaining tumor phenotype and survival of already transformed pheochromocytoma cells and is required for the initial establishment of these tumors, providing encouragement to carry on research studies to address the IGF-I/IGF-I receptor system as a target of therapeutic strategies for pheochromocytoma treatment in the future.

We read with interest the recent article by Hero et al. (1Hero M. Tommiska J. Vaaralahti K. et al.Circulating antimüllerian hormone levels in boys decline during early puberty and correlate with inhibin B.Fertil Steril. 2012; 97: 1242-1247Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) showing that antimüllerian hormone (AMH) is one of the earliest signs of pubertal development in healthy boys, occurring before any notable increase in testis volume or serum T. Owing to its longitudinal design, this study provides conclusive evidence for similar observations made in previous cross-sectional studies, indicating that the early increase in intratesticular T is responsible for the inhibition of AMH expression (2Rey R. Lordereau-Richard I. Carel J.C. et al.Anti-müllerian hormone and testosterone serum levels are inversely related during normal and precocious pubertal development.J Clin Endocrinol Metab. 1993; 77: 1220-1226Crossref PubMed Scopus (104) Google Scholar, 3Aksglæde L. Sorensen K. Boas M. et al.Changes in anti-Mullerian hormone (AMH) throughout the life span: a population-based study of 1027 healthy males from birth (cord blood) to the age of 69 years.J Clin Endocrinol Metab. 2010; 95: 5357-5364Crossref PubMed Scopus (183) Google Scholar, 4Grinspon R.P. Bedecarrás P. Ballerini M.G. et al.Early onset of primary hypogonadism revealed by serum anti-Mullerian hormone determination during infancy and childhood in trisomy 21.Int J Androl. 2011; 34: e487-e498Crossref PubMed Scopus (63) Google Scholar). The authors conclude from their results that Sertoli cells must begin to express the androgen receptor before the clinical onset of puberty. This does not contradict what was reported by Boukari et al. (5Boukari K. Meduri G. Brailly-Tabard S. et al.Lack of androgen receptor expression in Sertoli cells accounts for the absence of anti-Müllerian hormone repression during early human testis development.J Clin Endocrinol Metab. 2009; 94: 1818-1825Crossref PubMed Scopus (128) Google Scholar), who did not report the existence of androgen receptor in peripubertal boys because their study only included fetal, newborn, and adult testis samples. Interestingly, a recent study of our group (6Chemes H.E. Rey R.A. Nistal M. et al.Physiologic androgen insensitivity of the fetal, neonatal, and early infantile testis is explained by the ontogeny of the androgen receptor expression in Sertoli cells.J Clin Endocrinol Metab. 2008; 93: 4408-4412Crossref PubMed Scopus (134) Google Scholar) described that androgen receptor expression is first observed in the nuclei of few Sertoli cells at the age of 5 months. Weak labeling can be seen in 2%–15% of Sertoli cells until 4 years of age and progressively increases to high levels of androgen receptor expression in more than 90% of Sertoli cell nuclei by the age of 8 years (Fig. 1). Thereafter, an intense signal is present in almost all Sertoli cells. The presence of androgen receptor in boys older than 8 explains the early pubertal decline of AMH induced by intratesticular T rise. Circulating antimüllerian hormone levels in boys decline during early puberty and correlate with inhibin BFertility and SterilityVol. 97Issue 5PreviewTo investigate peripheral levels of inhibin B and antimüllerian hormone (AMH) in boys during peripuberty and in patients with congenital hypogonadotropic hypogonadism (HH). Full-Text PDF

ObjectiveTo clarify the physiopathological mechanisms of the acrosome and perinuclear theca development (PT) in Globozoospermia.DesignDescriptive.Materials and methodsSemen samples (5 Globozoospermia and 6 fertile) and germ cells from testicular biopsies (1 Globozoospermia and 3 with obstructive azoospermia with complete spermatogenesis) were included. Samples were evaluated by transmission electron microscopy (diaminobenzidine pre-embedding and immunogold labelling), immunofluorescence and western blot. For immunodetection, acrosin (C5F10), tubulin αβ and perinuclear theca (PT427) were detected by monoclonal antibodies. Six semen samples (3 fertile and 3 Globo) were used for western blot procedure; equal sperm proteins amounts (from 1.5 sperm million) were loaded onto a 10% SDS-PAGE. Statistical analysis was performed by T-test (P<0.05).ResultsIn normal spermiogenesis, acrosomal development depends on proper proacrosomal vesicle formation and simultaneous spermatid nuclear envelope modifications. TP was consistently immunolocalized in proacrosomal membrane vesicles and below the acrosome during its formation and expansion over the nucleus. In fertile men the TP is composed of six proteins (16-22-29-34-50-68 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.ConclusionThe alterations observed during early acrosome biogenesis in Globozoospermia are due to anomalous development of Golgi-derived proacrosomic vesicles and a significant deficiency of PT proteins. These findings demonstrate a key role of PT for proper acrosome formation, implatation and expansion over the spermatid nucleus. ObjectiveTo clarify the physiopathological mechanisms of the acrosome and perinuclear theca development (PT) in Globozoospermia. To clarify the physiopathological mechanisms of the acrosome and perinuclear theca development (PT) in Globozoospermia. DesignDescriptive. Descriptive. Materials and methodsSemen samples (5 Globozoospermia and 6 fertile) and germ cells from testicular biopsies (1 Globozoospermia and 3 with obstructive azoospermia with complete spermatogenesis) were included. Samples were evaluated by transmission electron microscopy (diaminobenzidine pre-embedding and immunogold labelling), immunofluorescence and western blot. For immunodetection, acrosin (C5F10), tubulin αβ and perinuclear theca (PT427) were detected by monoclonal antibodies. Six semen samples (3 fertile and 3 Globo) were used for western blot procedure; equal sperm proteins amounts (from 1.5 sperm million) were loaded onto a 10% SDS-PAGE. Statistical analysis was performed by T-test (P<0.05). Semen samples (5 Globozoospermia and 6 fertile) and germ cells from testicular biopsies (1 Globozoospermia and 3 with obstructive azoospermia with complete spermatogenesis) were included. Samples were evaluated by transmission electron microscopy (diaminobenzidine pre-embedding and immunogold labelling), immunofluorescence and western blot. For immunodetection, acrosin (C5F10), tubulin αβ and perinuclear theca (PT427) were detected by monoclonal antibodies. Six semen samples (3 fertile and 3 Globo) were used for western blot procedure; equal sperm proteins amounts (from 1.5 sperm million) were loaded onto a 10% SDS-PAGE. Statistical analysis was performed by T-test (P<0.05). ResultsIn normal spermiogenesis, acrosomal development depends on proper proacrosomal vesicle formation and simultaneous spermatid nuclear envelope modifications. TP was consistently immunolocalized in proacrosomal membrane vesicles and below the acrosome during its formation and expansion over the nucleus. In fertile men the TP is composed of six proteins (16-22-29-34-50-68 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. In normal spermiogenesis, acrosomal development depends on proper proacrosomal vesicle formation and simultaneous spermatid nuclear envelope modifications. TP was consistently immunolocalized in proacrosomal membrane vesicles and below the acrosome during its formation and expansion over the nucleus. In fertile men the TP is composed of six proteins (16-22-29-34-50-68 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. ConclusionThe alterations observed during early acrosome biogenesis in Globozoospermia are due to anomalous development of Golgi-derived proacrosomic vesicles and a significant deficiency of PT proteins. These findings demonstrate a key role of PT for proper acrosome formation, implatation and expansion over the spermatid nucleus. The alterations observed during early acrosome biogenesis in Globozoospermia are due to anomalous development of Golgi-derived proacrosomic vesicles and a significant deficiency of PT proteins. These findings demonstrate a key role of PT for proper acrosome formation, implatation and expansion over the spermatid nucleus.

Objective: To study expression of dysadherin in human testis, epididymis, and spermatozoa.Design: Prospective study.Setting: Basic research laboratory.Patient(s): Testis, epididymis, and testicular spermatozoa from patients under treatment and semen from volunteer donors.Intervention(s): Reverse transcription-polymerase chain reaction, immunohistochemistry, immunocytochemistry, and Western immunoblotting.Main Outcome Measure(s): Dysadherin messenger RNA (mRNA) analysis in testis, epididymis, and ejaculated spermatozoa, immunohistochemistry of both tissues, Western immunoblotting of tissue/cell extracts, and immunocytochemistry of spermatozoa.Result(s): Dysadherin mRNA was found in testis, epididymis, and ejaculated spermatozoa. Whereas testis and spermatozoa exhibited a distinctive 91-kDa protein form, the epididymis showed a 50-kDa moiety, also found in MDA-MB-231 breast cancer cells. Nucleotide sequence analysis revealed >99% homology between testicular and somatic cell mRNA, suggesting differential protein glycosylation. Dysadherin was immunodetected in round spermatids and testicular/ejaculated spermatozoa. It localizes to the acrosomal region and flagellum and colocalized with E-cadherin in the head and with the Na+,K+-ATPase alpha 4 subunit in the flagellum.Conclusion(s): This is the first report on expression of dysadherin in the male gonad and in spermatozoa. Its colocalization with E-cadherin and Na+, K+-ATPase leads us to postulate a role for dysadherin as a modulator of sperm function. (Fertil Steril (R) 2011;96:554-61. (C) 2011 by American Society for Reproductive Medicine.)