Exome sequencing reveals variants in known and novel candidate genes for severe sperm motility disorders.

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. STUDY FUNDING/COMPETING INTEREST(S): This project was supported in part by funding from the Australian National Health and Medical Research Council (APP1120356) to M.K.O.B., J.A.V. and R.I.M.L., The Netherlands Organisation for Scientific Research (918-15-667) to J.A.V., the Royal Society and Wolfson Foundation (WM160091) to J.A.V., as well as an Investigator Award in Science from the Wellcome Trust (209451) to J.A.V. and Grants from the National Research Council of Argentina (PIP 0900 and 4584) and ANPCyT (PICT 9591) to H.E.C. and a UUKi Rutherford Fund Fellowship awarded to B.J.H.

Exome sequencing reveals novel causes as well as new candidate genes for human globozoospermia.

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.

Is a CIS phenotype apparent in children with Disorders of Sex Development? Milder testicular dysgenesis is associated with a higher risk of malignancy.

All 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 pathology of the sperm centriole responsible for defective sperm aster formation, syngamy and cleavage.

BACKGROUND: In the present report we analyse the structural and functional features of sperm from a patient with severe asthenoteratozoospermia and failure of cleavage after ICSI. METHODS: Sperm were studied by phase contrast and transmission electron microscopy and microinjected into bovine oocytes to examine aster formation using antibodies against acetylated alpha- and beta-tubulins. RESULTS: Acephalic sperm, headless tails and abnormal alignments of the head-tail junction were observed. Flagella evidenced the features of dysplasia of the fibrous sheath. Bovine oocytes injected with patient's sperm showed male and female pronuclei but a faulty development of microtubules from the sperm-derived centrosome. The first ICSI attempt using conventional sperm selection methods resulted in fertilized two pronuclei zygotes, but no syngamy or cleavage. Three more ICSI attempts were performed, carefully avoiding sperm with obvious anomalies of the connecting piece. Fertilization and cleavage took place in all cycles, and in two of them positive betahCG plasma levels were detected but preclinical abortions ensued. CONCLUSIONS: We propose that the alterations in the head-tail junction and attachment, responsible for the observed sperm phenotype, result from centriolar dysfunctions that cause insufficient sperm aster formation, lack of syngamy and cleavage or defective embryos leading to early abortions.

Sperm ubiquitination in patients with dysplasia of the fibrous sheath.

BACKGROUND: Human sperm with structural abnormalities display an increased content of the cellular proteolytic marker peptide, ubiquitin. We investigated whether dysplasia of the fibrous sheath (DFS), a severe structural anomaly found in the sperm of some asthenozoospermic patients, is accompanied by (i) increased ubiquitination of the sperm surface and (ii) by increased ubiquitination of the sperm mitochondria. METHODS AND RESULTS: Five DFS patients and eight fertile donors were studied by immunocytochemistry with anti-ubiquitin antibodies. Increased cross-reactivity of the ubiquitinated mitochondrial epitopes was seen in 32-50% of DFS sperm, but only 2-4.1% of sperm from fertile donors. Sperm surface ubiquitination assessed by sperm-ubiquitin tag immunoassay (SUTI) and immunofluorescence demonstrated an increased sperm ubiquitination in all DFS patients. The average median value of ubiquitin-induced fluorescence in DFS patients was 25.8 counts (range 19.8-37.9), as opposed to 13.4 counts range (9.3-16.6) in fertile men. Sperm with 'stump tails', coiled tails, twin and triplet sperm, and clusters of immature spermatogenic cells were common. CONCLUSIONS: DFS sperm have increased cross-reactivity to anti-ubiquitin antibodies, a finding consistent with the ubiquitination of defective sperm shown in animal models. These results justify the use of ubiquitin-based assays for objective semen analysis in infertile men with heritable defects.

Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa.

Dysplasia of the fibrous sheath (DFS) is an anomaly found in spermatozoa of severe asthenozoospermic patients. Marked hypertrophy and hyperplasia of the fibrous sheath is the common characteristic. Immunocytochemistry allowed us to visualize the distortions and incidence of tail structure abnormalities associated with this phenotype in six patients; four with a complete form and two with an incomplete form of this pathology previously diagnosed and studied by electron microscopy. Microtubules and fibrous sheaths were studied using monoclonal antibodies against alpha-acetylated tubulin and anti-FSC1 (the major protein component of the fibrous sheath). Mitochondrial sheaths were visualized using the mitochondrion-specific vital dye MitoTracker green FM(TM). Phase contrast and fluorescent microscopy of semen samples showed large numbers of spermatozoa with short, rigid, thick and irregular tails. As expected, anomalous and completely distorted fibrous sheaths, severe alterations of the axonemal microtubules and different patterns of mitochondrial sheath configurations were found. While ultrastructural studies of thin sections allow an in-depth knowledge of the internal organization of the sperm tail, fluorescence labelling of selected sperm components affords a unique view of the whole flagellum including topographical relationships of various organelles. The combination of these different approaches is essential for a comprehensive understanding of this particular pathology.

Molecular genetic analysis of two human sperm fibrous sheath proteins, AKAP4 and AKAP3, in men with dysplasia of the fibrous sheath.

Dysplasia of the fibrous sheath (DFS) is characterized by male infertility, asthenozoospermia, and morphologically abnormal flagella that possess a severely malformed fibrous sheath. In many cases, DFS is familial, suggesting a genetic component. Human AKAP4 and AKAP3 are structural proteins of the fibrous sheath that also function to anchor protein kinase A to this structure via the regulatory subunit of the kinase. We hypothesized that defects in either AKAP4 or AKAP3 might cause DFS. No quantitative or qualitative differences between patients with DFS and normal controls were detected when sperm proteins were analyzed by either silver staining or immunoblot analysis using antibodies raised against AKAP4 and AKAP3. Additionally, AKAP4 and AKAP3 from DFS sperm retained the ability to bind the regulatory subunit of protein kinase A. Localization at the light and electron microscopic levels showed that AKAP3 and AKAP4 localized correctly to the FS of the amorphous flagellum in DFS sperm. Partial sequence analysis of the AKAP4 and AKAP3 genes in patients with DFS did not identify any significant alterations in potential AKAP4/AKAP3 binding regions, suggesting that the two proteins interact normally in DFS sperm. Our results did not find evidence to support the hypothesis that mutations in either gene are responsible for DFS in humans.

Infancy is not a quiescent period of testicular development.

Postnatal evolution of the testis in most laboratory animals is characterized by the close continuity between neonatal activation and pubertal development. In higher primates, infancy, a long period of variable duration, separates birth from the beginning of puberty. This period has been classically considered as a quiescent phase of testicular development, but is actually characterized by intense, yet inapparent activity. Testicular volume increases vigorously shortly after birth and in early infancy due to the growth in length of seminiferous cords. This longitudinal growth results from active proliferation of infantile Sertoli cells which otherwise display a unique array of functional capabilities (oestrogen and anti-müllerian hormone secretion, increase of FSH receptors and maximal response to FSH). Leydig cells also show recrudescence after birth, possibly determined by an active gonadotrophic-testicular axis which results in increased testosterone secretion of uncertain functional role. This postnatal activation slowly subsides during late infancy when periodic phases of activation of the hypothalamo-pituitary-testicular axis are paralleled by incomplete spermatogenic spurts. The beginning of puberty is marked by the simultaneous reawakening of Leydig cell function and succeeding phases of germ cell differentiation/degeneration which ultimately lead to final spermatogenic maturation. The marked testicular growth in this stage is due to progressive increase at seminiferous tubule diameter. Sertoli cells, which have reached mitotic arrest, develop and differentiate, establishing the seminiferous tubule barrier, fluid secretion and lumen formation, and acquiring cyclic morphological and metabolic variations characteristic of the mature stage. All of these modifications indicate that, far from being quiescent, the testis in primates experiences numerous changes during infancy, and that the potential for pubertal development and normal adult fertility depends on the successful completion of these changes.

Pregnancies established through intracytoplasmic sperm injection (ICSI) using spermatozoa with dysplasia of fibrous sheath.

AIM: Dysplasia of the fibrous sheath (DFS) is an anomaly found in asthenozoospermic patients with extremely low or absent motility. In order to determine the efficacy of ICSI in these patients, a retrospective analysis of ICSI results in DFS patients has been done. METHODS: Ten ICSI attempts were performed in 6 patients with diagnosis of Dysplasia of the Fibrous Sheath studied by transmission and scanning electron microscopy. RESULTS: In the cases studied, sperm concentration was (29.62 +/- 18.05) x 10(6)/mL, total motility was 1.14 +/- 1.31%. Progressive motility was 0% except for one case with 0.1% . One hundred and three preovulatory oocytes were obtained and 94 metaphase II oocytes were injected. Sixty-nine of them showed two pronuclei (fertilization rate: 73.4%). Forty-nine embryos were obtained and 34 were transferred (mean: 3.4 embryos per transfer). Five pregnancies were diagnosed by beta-hCG plasma level determinations that resulted to be one preclinical abortion, one clinical abortion and three deliveries. Another pregnancy (ongoing) was achieved from a cryopreserved embryo transfer. CONCLUSION: These results showed that ICSI provides a suitable solution for patients suffering from irreversible sperm defects such as DFS. Nevertheless, it is mandatory to inform couples of possible transmission risks to offspring, which are unknown at present. Only when the etiology of this problem is disclosed, it will be possible to assess the real genetic risk.

Acephalic spermatozoa and abnormal development of the head-neck attachment: a human syndrome of genetic origin.

A series of 10 young sterile men with acephalic spermatozoa or abnormal head-mid-piece attachments is presented. Nine of these patients had 75-100% spermatozoa with minute cephalic ends and 0-25% abnormal head-middle piece attachments. Loose heads ranged between 0-35 for each 100 spermatozoa and normal forms were rare. Two patients were brothers. On ultrastructural examination, the head was generally absent and the middle piece was covered by the plasma membrane. When present, heads implanted at abnormal angles on the middle piece. A testicular biopsy showed abnormal spermiogenesis. The implantation fossa was absent and the flagellar anlage developed independently from the nucleus, resulting in abnormal head-middle piece connections. In one patient azoospermia was induced with testosterone to attempt to increase the normal sperm clone during the rebound phenomenon, but all newly formed spermatozoa were acephalic. In another patient with high numbers of defective head-mid-piece connections, microinjections of spermatozoa resulted in four fertilized oocytes, but syngamy and cleavage did not take place, suggesting an abnormal function of the centrioles. The findings indicate that acephalic spermatozoa arise in the testis as the result of an abnormal neck development during spermiogenesis. The familial incidence and the typical phenotype strongly suggest a genetic origin of the syndrome.

A novel E153X point mutation in the androgen receptor gene in a patient with complete androgen insensitivity syndrome.

AIM: To study a 46, XY newborn patient with a phenotype suggestive of an androgen insensitivity syndrome to confirm an anomaly in the AR gene. METHODS: Genomic DNA from leukocytes was isolated in order to analyze SRY gene by PCR and sequencing of the eight exons of AR gene. Isolation of human Leydig cell mesenchymal precursors from the testis was performed in order to study testosterone production and response to hCG stimulation in culture. RESULTS: Surgical exploration disclosed two testes, no Wolffian structures and important Müllerian derivatives. The SRY gene was present in peripheral blood leukocytes. Sequencing of the AR gene evidenced a previously unreported G to T transversion in exon 1 that changed the normal glutamine 153 codon to a stop codon. Interstitial cell cultures produced sizable amounts of testosterone and were responsive to hCG stimulation. CONCLUSION: This E153X nonsense point mutation has not been described previously in cases of AIS, and could lead to the synthesis of a short truncated (153 vs 919 residues) non functional AR probably responsible for the phenotype of complete androgen insensitivity syndrome (CAIS).

Ultrastructural pathology of the sperm flagellum: association between flagellar pathology and fertility prognosis in severely asthenozoospermic men.

An ultrastructural study of spermatozoa in a series of 247 severely asthenozoospermic patients disclosed two kinds of anomalies. The first was dysplasia of the fibrous sheath, a primary defect of spermatozoa with hypertrophy and hyperplasia of the fibrous sheath, associated axonemal anomalies, familial incidence and chronic respiratory disease. The patients could be divided into two subgroups: the complete form (all spermatozoa affected) and the incomplete form (alterations in 70-80% spermatozoa). There were no spontaneous or in-vitro fertilization (IVF) pregnancies. Intracytoplasmic sperm injection (ICSI) in six patients resulted in successful fertilizations, but only two pregnancies were obtained. These features configure a phenotype that suggests a genetic origin. The second anomaly was non-specific flagellar anomaly (NSFA), random secondary flagellar alterations affecting variable numbers of spermatozoa, without respiratory disease or familial incidence. 54 men with NSFA were followed for 2-6 years. Of these, 18 achieved conception, either spontaneous or by means of assisted fertilization, followed by 14 pregnancies and 12 live births. Their sperm motility significantly increased during the follow-up period. In the remaining 36 men motility did not change during the follow-up period and there were no fertilizations or pregnancies. We conclude that in severe asthenozoospermia, ultrastructural examination of spermatozoa has an effective prognostic value, identifying two syndromes with very different flagellar alterations and fertility potentials.

Successful intracytoplasmic sperm injection with spermatozoa from a patient with dysplasia of the fibrous sheath and chronic respiratory disease.

The present report describes a successful intracytoplasmic sperm injection (ICSI) procedure performed with immotile spermatozoa from a young man with a combination of dysplasia of the fibrous sheath and dynein deficiency, a recently described variant of the immotile cilia syndrome. This methodology provides the only suitable solution for these patients in whom all other assisted fertilization technologies have previously failed, and opens the possibilities for treatment of male infertility due to severe, irreversible sperm defects such as the one reported here.

Is infancy a quiescent period of testicular development? Histological, morphometric, and functional study of the seminiferous tubules of the cebus monkey from birth to the end of puberty.

The objective of this study was to describe the maturational changes observed in the seminiferous tubules of the monkey Cebus apella, a New World primate species, from birth to the end of puberty. Nineteen animals were subdivided into four groups: neonatal (1-40 days), infantile (4 months to 1 yr), early pubertal (1 yr, 8 months to 2 yr, 9 months), and late pubertal (4-8 yr). Volumetric determinations of different testicular components were made, tubule diameter and length were calculated, and spermatogenic cells, Sertoli cells, and androgen-binding protein secretion were quantified. Testicular and seminiferous tubule volumes increased significantly in the first 5 months of life and during puberty due to the combined increment in seminiferous tubule diameter and length. The total number of spermatogonia increased until late puberty to stabilize subsequently. Spermatocytes and spermatids appeared during puberty and increased dramatically until the end of this period. The germ cell ratios, indicative of spermatogenic efficiency, improved continuously in late puberty coincidentally with a reduction of spermatocyte degeneration. Sertoli cells proliferated in the neonatal and infantile periods, determining a longitudinal growth of the seminiferous tubules, but remained stable during puberty, when androgen-binding protein secretion increased significantly. The multiplication of germ cells is the main factor responsible for the increment in tubule diameter during puberty and determines the most noticeable postnatal modification of testicular volume. During late puberty, the reduction of spermatocyte degeneration leads to an increment in germ cell ratios and a progressive, but slow, improvement of spermatogenic efficiency, explaining why pubertal development of the testis occurs over such a prolonged period in this primate. This is in contrast to what happens in most laboratory animals and suggests that the Cebus is a useful model for studies of human male puberty.

Extreme asthenozoospermia and chronic respiratory disease: a new variant of the immotile cilia syndrome.

Two patients suspected of suffering from ciliary dyskinesis were investigated. They consulted for primary infertility and chronic respiratory disease. Functional lung studies showed obstructive changes in one patient. Both had immotile sperm with short, thick and rigid tails. Ultrastructural studies of nasal biopsies showed abnormal cilia with almost complete lack of inner dynein arms (mean number of inner arms per axoneme 0.67 +/- 1.21 in patient 1 and 1.49 +/- 1.17 in patient 2, compared with normal values of 5.3 +/- 0.13). Other abnormalities included lack of parallel orientation of cilia and central translocation of microtubular doublets. Electron microscopy of sperm revealed hyperplasia of the fibrous sheath and axonemal disruption. This is the first report of an association of different anomalies in cilia and flagella leading to clinical manifestation of the immotile cilia syndrome. These findings emphasize the need for ultrastructural examination of respiratory cilia in men suffering from fibrous sheath alterations of sperm which so far have not been described in patients with the classical form of immotile cilia syndrome.

Developmental changes in testicular interstitial cell populations, LH receptors and in the response to hCG in the rat.

High doses of hCG were administered to immature rats of different ages and the animals killed 48 h later. Serum testosterone increased 2 to 4-fold over control values 48 h after hCG. In-vitro androgen production showed different patterns according to age. Animals younger than 35 days, when treated with hCG, retained the ability to respond to in-vitro gonadotrophic stimulation. This ability was lost in testes from rats aged 45 days. The number of free LH-receptors 48 h after hCG diminished with increasing age to become non-detectable at 35 and 45 days. In control animals the proportion of differentiated Leydig cells in relation to their mesenchymal precursors increased progressively with age to reach highest values at 45 days. hCG administration induced a shift of the cellular composition of the interstitium toward the more mature cell types. hCG has a predominantly trophic action on mesenchymal precursors in young rats, promoting their differentiation. These effects are minimal in the differentiated Leydig cells in older animals. It is proposed that the observed biochemical responses are the result of the balance between the increase in LH receptors and steroidogenic enzymes in the developing new generation of young Leydig cells and the down-regulation of receptors and enzymatic lesions in fully differentiated Leydig cells.

Dysplasia of the fibrous sheath: an ultrastructural defect of human spermatozoa associated with sperm immotility and primary sterility.

A study of a group of five patients presenting with primary sterility and showing severe sperm immotility is presented. Most spermatozoa in these patients showed rigid, short, thick, and/or irregular tails and 95 to 100% were immotile. Electron-microscopy disclosed a common pattern of flagellar abnormalities. There was a dysplastic development of the fibrous sheath, which appeared hyperplastic and disorganized. The axoneme was either missing or grossly distorted. In a few instances, a normal flagellum could be identified. Similar alterations also were detected in maturing spermatids, suggesting that the described defect develops during spermiogenesis. Two of the five patients had recurrent bronchial and sinusal infections and bronchiectasis, suggesting the possible existence of an associated abnormality in respiratory cilia. The existence of a common ultrastructural defect affecting most spermatozoa, its presence in two brothers, and the possibility of association with immotile respiratory cilia point to the existence of a syndrome (namely the "dysplasia of the fibrous sheath") of possible familial transmission.

Lack of a head in human spermatozoa from sterile patients: a syndrome associated with impaired fertilization.

Three patients with primary sterility in whom the majority of spermatozoa lacked a normally implanted head are presented. A small cephalic knob was evident in most of them by routine colorimetric techniques, and the Feulgen reaction failed to show any deoxyribose nucleic acid. The morphologic features of the tails was normal. Few loose sperm heads were observed in the ejaculates. Even though motility was decreased, there were numerous acephalic sperms with different degrees of forward motility. Electron microscopy showed a well-organized structure of the centrioles and connecting piece, which were located in the neck region within a small cytoplasmic mass, but no chromatin was detected in any case. Studies on immature spermatids present in semen evidenced an independent anomalous development of heads and tails and suggested that they became separated at the end of spermatid maturation. This anomaly, of probable genetic origin, is interpreted to be due either to an alteration in the mechanism of migration and positioning of the tail on the caudal pole of the nucleus or to an interference with the formation of the implantation fossa of the head, which normally accommodates the connecting piece.

Acute hCG administration induces seminiferous tubule damage in the adult rat.

In order to study the early (or short time) effects of an hCG stimulation on the seminiferous tubules of the adult rat, a single dose of 100, 200 or 400 IU hCG was administered to eighty to ninety day old rats of the Sherman strain. The histological analysis revealed a tubular damage already noticeable six hours after the injection. This precocious lesion becomes more pronounced two to five days later, consisting of degeneration and hypocellularity of the germinal epithelium, margination of the chromatin in round spermatids and formation of multinuclear giant cells. This damage involves big areas of the testis, particularly of peripheral location. The analysis performed three months after the acute stimulation still showed tubular regressive changes, thus indicating an incomplete reversion of the damage. Changes in the hormonal environment of hCG treated rats were found. Serum testosterone significantly increased from 6 to 72 hours following a 200 IU hCG injection. A severe diminution of serum FSH levels and a significant increase of the serum estradiol were observed. Intratesticular administration of estradiol benzoate was able to reproduce the tubular damage in some animals, while restoration of FSH circulating levels by simultaneous administration of hCG and purified hFSH did not prevent the changes induced by hCG alone. These results suggest that the high intratesticular level of estradiol and not the lowering of serum FSH might be the mechanism responsible for the described testicular injury.