Immature Testicular Tissue Research Articles

Vitrification of non-human primate immature testicular tissue allows maintenance of proliferating spermatogonial cells after xenografting to recipient mice

This study demonstrates preservation of tissue integrity, maintenance of proliferating spermatogonia and Leydig cell functionality after vitrification and transplantation of non-human primate immature testicular tissue. The objective was to assess the potential of vitrification of non-human primate immature testicular tissue (ITT) in an in vivo xenotransplantation model. Testicular tissue was obtained from one immature rhesus monkey (Macaca mulatta) aged 4 years. Collection and vitrification of testicular tissue, followed by short-term xenografting (3 wks) to nude mice were performed to evaluate and compare vitrified/warmed and fresh tissue. Fresh ungrafted tissue was used for control purposes. Cell density and seminiferous tubule (ST) integrity were assessed by light microscopy. Presence of spermatogonia (SG) (MAGE-A4), proliferation (Ki-67) and Leydig cell (LC) functionality (3β-hydroxysteroid dehydrogenase; 3β-HSD) were evaluated by immunohistochemistry (IHC). Qualitative analysis revealed preservation of the histologic characteristics of SG and Sertoli cells (SCs), as well as cell-cell cohesion and cell adhesion to the basement membrane, in both vitrified and fresh grafted tissues. Survival of SG able to proliferate and functional LCs was confirmed by IHC in fresh and vitrified grafts. In conclusion, vitrification appears to be a promising approach, representing an alternative strategy to slow-freezing in the emerging field of ITT cryopreservation and cryobanking.

Efficient cryopreservation of testicular tissue: effect of age, sample state, and concentration of cryoprotectant

To study the effect of age and sample state on cryopreservation of testicular tissue, evaluate toxicity of commonly used cryoprotectants (CPs), and determine their optimal concentration for use. Prospective experimental study. Academic research unit. Patients with prostate carcinoma undergoing orchidectomy. We also studied immature and adult male Holtzman rats. Toxicity of CPs before freezing, morphology, and relative viability after freezing were evaluated for rat testicular cell suspensions (CS) and tubular fragments (TUB). Relative viability of adult human testicular CS and TUB after thaw was evaluated. Human TUB were cultured after thaw for 48 hours in medium containing epidermal growth factor (EGF), and effects on viability, morphology, and gene expression were determined. Viability and ploidy were measured with flow cytometry, postthaw cryodamage of immature rat tissue was studied by transmission electron microscopy, cell proliferation and differentiation were evaluated by immunohistochemistry and by real-time polymerase chain reaction. Immature testicular tissue was more susceptible to toxic assault by CP than adult tissue and displayed cell-specific sensitivity to CP, with glycerol, dimethyl sulfoxide and ethylene glycol being effective in protecting spermatid (1N), spermatogonia (2N) and spermatocyte (4N) populations respectively. Preservation as TUB may be preferred over CS and DMSO is an effective CP for immature and ethylene glycol for adult testicular tissue. Differential sensitivity of immature testicular tissue to CPs warrants judicious selection of CP on the basis of end application for prepubertal tissue.

Assessment of freezing procedures for rat immature testicular tissue

Fertility preservation has been included in the management of childhood cancer treatment. Cryopreservation of immature testicular tissue is the only available solution for pre-pubertal boys. Different freezing protocols have been developed in several species but without a clearly identified procedure. We tried to evaluate several protocols for cryopreservation of rat immature testicular tissue. Twelve different freezing protocols using different (i) cryoprotectant (dimethylsulphoxide [DMSO] or 1,2-propanediol [PROH]), (ii) cryoprotectant concentration (1.5M or 3M), (iii) equilibration time (30 or 60 min), (iv) equilibration temperature (4 °C or room temperature), (v) size of testicular fragment (7.5mg or 15mg), (vi) package (straws or cryovials), were compared using cord morphological damage evaluation. A testicular tissue piece of 7.5mg cryopreserved in cryovial using 1.5M DMSO, an equilibration time of 30 min at 4 °C showed fewer morphological alterations than the other protocols tested. The selected freezing protocol was able to maintain rat immature testicular tissue architecture, functionality after testicular pieces organotypic culture, and could be proposed in a human application.

Préservation de la fertilité chez l’enfant prépubère

Gonadotoxic therapies during childhood may impair future fertility in adult life and fertility preservation techniques should be discussed before starting gonadotoxic therapies. In both sexes, fertility preservation often means immature gametes cryopreservation. For girls, ovarian tissue cryopreservation is the only existing option to preserve fertility in prepubertal girls at risk of premature ovarian failure. This promising approach involves the storage of a large number of follicles, which could subsequently be transplanted or cultured to obtain mature oocytes. The results of ovarian tissue cryopreservation in adults are encouraging. At least nine children have been born after orthotopic reimplantation of frozen-thawed ovarian cortex. None of these pregnancies were obtained by reimplantation of ovarian tissue harvested before puberty; however, the probability of restoring fertility should be higher for younger girls, as their ovarian cortex clearly contains a large number of follicules. In vitro growth of primordial follicles to mature oocytes could be an option but this goal has not yet reached in humans. This option may be reach in the future, when young patients are in their twenties or thirties. For boys, spermatogonial stem cells can be cryopreserved and uni or bilateral testicular pieces can be stored for future use. Animal data reveals that healthy offspring were reported after grafting of frozen testicular cell suspensions or tissue pieces in different species. Although recent data show promising results, restoring fertility by using frozen testicular cells after transplantation or in vitro culture is not shown yet. Then, immature testicular tissue cryopreservation for prepubertal boys is still an experimental procedure. However, as their use for restoring fertility should not be requested before 10-30 years, a long time is given for advances in medical research.

Can prepubertal human testicular tissue be cryopreserved by vitrification?

To assess vitrification of prepubertal human testicular tissue in vitro. Case report. Academic research unit. Two patients (6 and 12 years of age) who were to start gonadotoxic treatment for chronic granulomatous disease and acute lymphoblastic leukemia. Long-term (10-day) organotypic culture performed immediately after vitrification and warming. Fresh tissue and tissue cryopreserved by slow-freezing were used as control samples. Spermatogonial cell survival (MAGE-A4) and proliferation (Ki67) were evaluated by immunohistochemistry (IHC) and tubular integrity by light microscopy. Qualitative analysis revealed that histologic characteristics of spermatogonia and Sertoli cells were preserved, as were cell-cell cohesion and cell adhesion to the basement membrane, in vitrified tissue as well as in frozen and fresh control samples. Survival of spermatogonia and their ability to proliferate as evidenced by IHC was also confirmed in cultured fresh, slow-frozen, and vitrified tissue. Vitrification, having the advantage of being a faster and more convenient method, shows promise as an alternative strategy to slow-freezing in the emerging field of immature testicular tissue cryopreservation.

Management of fertility preservation in prepubertal patients: 5 years’ experience at the Catholic University of Louvain

BACKGROUND Since prepubertal boys cannot benefit from sperm banking, a potential alternative strategy for fertility preservation involves immature testicular tissue (ITT) banking aimed at preservation of spermatogonial stem cells. Survival of spermatogonia has been demonstrated after ITT freezing, which is considered ethically acceptable. We report the results of a pilot program set up for fertility preservation in prepubertal boys. METHODS All boys undergoing ITT cryobanking from May 2005 were identified from our clinical register. Data were collected from medical files. RESULTS Testicular tissue was retrieved from 52 prepubertal patients under 12 years of age and 10 peripubertal patients aged between 12 and 16 years, in whom no spermatozoa were identified in testicular biopsies. Malignant disease accounted for 80.6% of cases; the remaining patients suffered from benign disorders requiring gonadotoxic treatments. Mean ages, Tanner stages and occurrence rates of urogenital pathology were 6.43 ± 3.32 and 14 ± 1.23 years, I and I-IV, and 13.5 and 20% for pre- and peripubertal patients, respectively. Mean volumes of removed tissue were 20.1 ± 8.6 and 42.4 ± 15.6 mm(3) for pre- and peripubertal patients, respectively. No complications occurred during or after tissue retrieval and 93.5% of referred patients accepted ITT storage. The presence of spermatogonia, and thus the potential for later tissue use, was established in all of these patients. CONCLUSIONS The majority of cryopreserved samples showed reproductive potential. Storage was accepted by most parents. All parents and children considered this fertility preservation strategy a positive approach.

Rapid Screening of Cryopreservation Protocols for Murine Prepubertal Testicular Tissue by Histology and PCNA Immunostaining

Numerous parameters have to be tested to identify optimal conditions for prepubertal testicular tissue banking. Our study evaluated 19 different cryopreservation conditions for immature testicular tissue using a rapid screening method. Immature mice testes were cryopreserved using either 1,2-propanediol (PROH) or dimethyl sulfoxide (DMSO) at a concentration of 0.75 or 1.5 M using a controlled slow-cooling rate protocol with (S+) or without seeding (S+). Equilibration was performed either at room temperature or at 4°C for 15 or 30 minutes. Seminiferous cord cryodamage was determined by scoring morphologic alterations. Cell proliferation ability was evaluated using a proliferating cell nuclear antigen (PCNA) antibody. Testes cryopreserved with optimal conditions were grafted into immunodeficient mice. The highest proportions of PCNA-positive nuclei and lowest morphologic alterations were observed with 1.5 M DMSO. Tissues were more altered with 0.75 M DMSO or PROH. Complete germ cell maturation was observed after allografting of testicular pieces previously frozen with 1.5 M DMSO, S+, 30 minutes. The 1.5 M DMSO, S+ or S+ protocol preserved prepubertal mice testicular tissue architecture and germ cell and Sertoli cell proliferation potential. Allografting of thawed testis fragments into immunodeficient mice confirmed that the 1.5 M DMSO, S+, 30 minutes protocol maintained testicular somatic and germ cell functions. Postthaw histologic evaluation and PCNA immunostaining are useful to rapidly test numerous freeze-thaw parameters. They may also be efficient tools to control human prepubertal frozen testis quality, within the context of a clinical application.

In Vitro Murine Spermatogenesis in an Organ Culture System1

Achieving mammalian spermatogenesis in vitro has a long history of research but remains elusive. The organ culture method has advantages over the cell culture method, because germ cells are in situ albeit the tissue as a whole is in vitro. The method was used in the 1960s and 1970s but encountered difficulties in inducing complete meiosis, i.e., in getting meiosis to proceed beyond the pachytene stage. In the present study, we reevaluated the organ culture method using two lines of transgenic mice, Acr-GFP and Gsg2 (haspin)-GFP mice, whose germ cells express green fluorescent protein (GFP) at the mid and end stages of meiosis onward, respectively. Immature testicular tissues from these mice, ranging from 4.5 to 14.5 days postpartum, were cultured on the surface of the medium, providing a liquid-gas interface. Culturing testicular tissues of all ages tested resulted in the expression of both Acr- and Gsg2-GFP. Round spermatids were identified by a combination of Gsg2-GFP expression, cell size, and the presence of a single nucleus with a dot stained by Hoechst. In addition, the chromosome number of one of such presumptive spermatids was found to be 20 by the premature chromosome condensation method. As our semiquantitative assay system using GFP expression grading was useful for monitoring the effects of different environmental factors, including temperature, oxygen concentration, and antiretinoic molecules, further improvement of the culture conditions should be possible in the future.

Donor-host involvement in immature rat testis xenografting into nude mouse hosts.

Immature testicular tissue of a wide variety of mammalian species continues growth and maturation when ectopically grafted under the dorsal skin of adult nude mouse recipients. Tissues from most donor species fully mature, exhibiting complete spermatogenesis within months. The connection to the recipient's vascular system is mandatory for graft development, and failure of vascularization leads to necrosis in the grafted tissue. In the present study, we analyze to what extent 1) the xenografted immature donor tissue and 2) the recipient's cells and tissues contribute to the functional recovery of a "testicular xenograft." We address whether recipient cells migrate into the testicular parenchyma and whether the circulatory connection between the donor testicular tissue and the recipient is established by ingrowing host or outgrowing donor blood vessels. Although this issue has been repeatedly discussed in previous xenografting studies, so far it has not been possible to unequivocally distinguish between donor and recipient tissues and thus to identify the mechanisms by which the circulatory connection is established. To facilitate the distinction of donor and recipient tissues, herein we used immature green fluorescent protein-positive rat testes as donor tissues and adult nude mice as graft recipients. At the time of graft recovery, donor tissues could be easily identified by the GFP expression in these tissues, allowing us to distinguish donor- and recipient-derived blood vessels. We conclude that the circulatory connection between graft and host is established by a combination of outgrowing small capillaries from the donor tissue and formation of larger vessels by the host, which connect the graft to subcutaneous blood vessels.

Options for fertility preservation in prepubertal boys

Fertility in adult life may be severely impaired by gonadotoxic therapies. For young boys who do not yet produce spermatozoa, cryopreservation of immature testicular tissue (ITT) is an option to preserve their fertility, albeit still experimental. This paper covers current options for ITT cryopreservation and fertility restoration. Relevant studies were identified by an extensive Medline search of English and French language articles. Search terms were: gonadotoxicity, cytoprotection, cryopreservation, ITT, spermatogonia, testicular transplantation, testicular grafting and in vitro maturation (IVM). Although no effective gonadoprotective drug is yet available for in vivo spermatogonial stem cell protection in humans, current evidence supports the feasibility of ITT cryopreservation before gonadotoxic treatment with a view to fertility preservation. Controlled slow freezing with dimethyl sulfoxide allows survival and proliferation of human spermatogonia after xenotransplantation, but only partial differentiation. Animal data look promising, since healthy offspring have been obtained after transplantation of frozen testicular cell suspensions or tissue pieces. However, none of the fertility restoration options from frozen tissue, i.e. cell suspension transplantation, tissue grafting and IVM have proved efficient and safe in humans as yet. While additional evidence is required to define optimal conditions for ITT cryopreservation with a view to transplantation or IVM, the putative indications for such techniques, as well as their limitations according to disease, are outlined.

Testicular stem cells for fertility preservation: Preclinical studies on male germ cell transplantation and testicular grafting

Spermatogonial stem cells open novel strategies for preservation of testicular tissue and fertility preservation in boys and men exposed to gonadotoxic therapies. This review provides an update on the physiology of spermatogonial stem cells in rodent and primate testes. Species-specific differences must be considered when new technologies on testicular stem cells are considered. Germ cell transplantation is presented as one novel and promising strategy. Whereas this technique has become an important research tool in rodents, a clinical application must still be regarded as experimental and many aspects of the procedure need to be optimized prior to a safe and efficient clinical application in men. Testicular grafting opens another exciting strategy for fertility preservation. Autologous and xenologous transfer of immature tissue revealed a high regenerative potential of immature testicular tissue. Grafting was applied in rodents and primates and resulted in the generation of sperm. Further research is needed before an application in humans can be considered safe and efficient. Despite the current limitations in regard to the generation of sperm from cryopreserved male germline cells and tissues, protocols for cryopreservation of testicular tissue are available and reveal a promising outcome. Since future improvements of germ cell transplantation and grafting approaches can be assumed, bioptic retrieval and cryopreservation of testicular tissue fragments should be performed in oncological patients at high risk of fertility loss since this is their only option to maintain their fertility potential.

Long-term spermatogonial survival in cryopreserved and xenografted immature human testicular tissue

Preservation of the male germ line in prepubertal boys undergoing gonadotoxic treatment is a crucial consideration in terms of their future quality of life. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival, proliferation and differentiation capacity of spermatogonia after cryopreservation and long-term transplantation of immature testicular tissue pieces. Single pieces of testicular tissue (2-8 mm(3)) from prepubertal boys (7-14 years) were cryopreserved, thawed and transplanted into the scrotum of mice for 6 months. Upon removal, histological, immunohistochemical and ultrastructural analyses and testicular sperm extraction (TESE) were used to evaluate the tissue. Histology showed 55 +/- 42% of tubules to be intact. MAGE-A4 immunostaining showed mean spermatogonial recovery to be 3.7 +/- 5.5%, with 35% of these cells expressing Ki67, evidencing proliferation in tissue from boys <14 years of age. No apoptosis was found, as demonstrated by the absence of active caspase-3 and TUNEL staining. Numerous premeiotic spermatocytes, a few spermatocytes at the pachytene stage and spermatid-like cells were observed. No immunostaining was observed for lactate dehydrogenase-C, ACE or proacrosin, indicating that the spermatid-like structures observed by histology did not express the meiotic and post-meiotic markers characteristic of normal spermatids. No ultrastructural alterations of the tissue were encountered. The present study demonstrates that spermatogonia are able to survive and proliferate after cryopreservation and long-term transplantation. Complete regeneration of normal spermatogenesis was not observed since, beyond the pachytene stage, no adequate characterization of germ cells was obtained. Further studies are thus required to investigate the differentiation potential of cryopreserved germ cells.

Donor/Host Involvement in Testis Xenografts.

When immature testicular tissue of a wide variety of mammalian species is ectopically grafted under the dorsal skin of adult nude mouse hosts, the grafted tissue continues to grow in the new location, and, in most donor species, fully matures, exhibiting complete spermatogenesis within months. Xenografted testicular tissue establishes a connection to the hosts circulatory system. It is usually enclosed in a sturdy capsule. Here we analyze, to what extent a) the xenografted immature donor tissue and b) the hosts cells and tissues contribute to the functional "testicular xenograft", and whether the circulatory connection between the donor testicular tissue and the host are established by ingrowing host or outgrowing donor blood vessels. For this purpose, we grafted a total of 78 halves of testes of 3-5 day GFP+ rats with a Sprague-Dawley background into 13 adult (10-12 weeks old) castrated nude mouse recipients. The grafts were allowed to develop for 4 weeks. 59 xenografts and adjacent tissues (capsule, connective tissue, blood vessels) were retrieved (76% recovery rate), with graft weights ranging from 17 to 711 mg (average weight 162 mg). GFP+ (=donor rat) and GFP- (=host mouse) tissues were then distinguished either by microscopic detection of native GFP-fluorescence on cryosections, or by detection of GFP using immunohistochemistry employing an anti-GFP primary antibody and fluorescent secondary antibodies on paraffin sections. Here we show that all tissues within the testicular parenchyma, including blood vessels, are exclusively of rat donor origin. The capsule surrounding the testicular parenchyma is composed of two distinct layers, the thinner inner one being of rat origin, and the thicker outer one being formed by the mouse host. All blood vessels emerging from the testicular parenchyma and protruding beyond the capsule show a GFP+ endothelium indicating that the connection between xenografted immature rat testicular tissue and the nude mouse hosts blood circulation is established by blood vessel growing out from the xenografted tissue. We conclude, that both donor and host are involved in encapsulating immature xenografted rat testicular tissue, but that the formation of a circulatory connection between xenograft and host is initiated and primarily executed by the donor tissue. Grant support: NIH 2U54 HD008610 and Lance Armstrong Foundation Young Investigator Award Issues of Cancer Survivorship

Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts

Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts

Endocrine Status and Development of Porcine Testicular Tissues in Host Mice

Clarification of the endocrine status of host mice provides us with basic knowledge with which we can manipulate the growth and function of xenografted testicular tissues. We investigated the hormonal profiles of castrated mice grafted with porcine immature testicular tissues from 30 to 210 or more days after grafting (day 0=castration and grafting). The serum follicle stimulating hormone (FSH) concentrations of the host mice declined (P<0.05) from day 60 compared with those of the castrated, ungrafted mice. The serum inhibin and testosterone levels were higher (P<0.05) than those in the castrated, ungrafted mice from days 30 and 90 days, respectively. The inhibin levels further increased (P<0.05) from day 90, during which time the levels were higher (P<0.05) than those in the intact male mice. In the grafts, formation of lumens occurred in the seminiferous cords on day 90 and spermatozoa appeared in the lumens from day 120. However, spermatogenesis in the grafts did not reach the qualitatively normal levels observed in adult boars. The intensity of the immune reaction to inhibin alpha subunits in the Sertoli cells of the grafts decreased with differentiation of the seminiferous tubules. The present findings indicate that a feedback loop was established between the mouse hypothalamo-pituitary axis and the grafted porcine tissues from 60 days post-grafting. The results also indicate that the serum inhibin levels in the host mice remained high even after the appearance of lumens in the seminiferous tubules of the grafted tissues; this is strikingly different to the situation in normal male animals, in which the serum inhibin levels decline at around the time of tubular differentiation. The lack of efferent ducts in the tubules of the grafted tissues probably caused the accumulation of inhibin to be released into the lumens, resulting in high concentrations of circulating inhibin. These high levels of inhibin may directly affect spermatogenic activity and suppress FSH secretion.

Comparison of conditions for cryopreservation of testicular tissue from immature mice

Cryopreservation of immature testicular tissue could be considered as a major step in fertility preservation for young boys with cancer. In the present study, eight different freezing protocols were evaluated in immature mice testis. Testis from six-day-old mice were frozen using either 1,2-propanediol (PROH) or dimethylsulphoxide (DMSO: D) at 1.5 M. Different cooling rate curves were tested: (i) controlled slow protocol with seeding (CS+) or (ii) without seeding (CS-), (iii) controlled rapid protocol and (iv) non-controlled protocol. Cryodamage of seminiferous cords was semi-quantitatively determined, establishing a scoring of alterations. Cell viability and apoptosis induction were assessed on testicular cell suspensions immediately after digestion (D0) and after a 20-h culture period (D1). Cells recovered after digestion of 100 mg tissue and the rate of living and non-apoptotic cells were quantified at D0 and D1. A long-term culture (9 days) of testis pieces was carried out for the protocol offering the best survival. Testosterone production, intratubular cell proliferation and tubule growth were assessed. DMSO produced optimal results in the different cooling rate curves tested when compared with PROH. Optimal results were obtained for the DCS- procedure (P < 0.05). Testosterone production, tubule growth and cell proliferation of post-thaw pieces were similar to fresh samples. Testis freezing with 1.5 M DMSO in a CS- procedure was found to maintain not only immature testicular tissue architecture, but also viability of testicular cells, endocrine and partial exocrine functions of the testis. Semi-quantitative evaluation of seminiferous cord cryodamage can be effectively used to rapidly screen optimal freezing conditions and as a possible quality control in a human application.

Spermatogonial survival after cryopreservation and short-term orthotopic immature human cryptorchid testicular tissue grafting to immunodeficient mice

Fertility preservation has become an urgent clinical requisite for prepubertal male cancer patients undergoing gonadotoxic treatment. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival and proliferative activity of spermatogonia and Sertoli cells after cryopreservation of cryptorchid testicular tissue pieces followed by xenografting for 21 days. Single pieces of tissue from cryptorchid testes (2-9 mm(3)) of young boys (2-12 years) were cryopreserved, thawed and transplanted into the scrotum of mice. Quantitative morphometric and immunohistochemical techniques were used to evaluate the integrity of the tissue, as well as the survival and proliferative capacity of spermatogonia and Sertoli cells before and after freezing/thawing/grafting. Three weeks after grafting, cryopreserved tissue was removed and analysed. Most of the tubules (88.3%) were intact and there was no fibrosis or sclerosis, 14.5% of the initial spermatogonial population remained, as identified by the MAGE A4 antibody, and 32% of these cells showed proliferative activity evidenced by Ki67, compared to 17.8% before cryopreservation and grafting. The number of Sertoli cells was unchanged and 5.1% were Ki67-positive, compared to none at all before freezing and grafting. Through our orthotopic xenografting model, we have demonstrated the survival and proliferative activity of spermatogonia and Sertoli cells in cryopreserved immature human cryptorchid tissue. Testicular tissue banking may thus prove to be a promising technique for the preservation of fertility in prepubertal boys undergoing oncological treatments. As the stem cell niche is maintained, the cryopreserved tissue can potentially be used for future autotransplantation. In addition, whole tissue freezing does not exclude alternative clinical uses, including isolated cell transplantation after dissociation, selection and enrichment. However, as this work was done on cryptorchid tissue, studies on normal immature testicular tissue, involving longer grafting periods, are needed to demonstrate a differentiation capacity before clinical implementation. Ethical and safety issues should also be addressed.

Distribution of sex chromosomes in dysgenetic gonads of mixed type

In a four-week-old child with female external and internal genitalia but with clitoris hypertrophy chromosome analysis from blood lymphocytes revealed a 46,XY karyotype. No deletion of Y chromosomal sequences was detected by PCR analysis of genomic DNA isolated from peripheral blood leucocytes. Because of the increased risk for gonadal tumours, gonadectomy was performed. Conventional cytogenetic analysis of the left dysgenetic gonad revealed a gonosomal mosaicism with a 45,X cell line in 27 of 50 metaphases. The dysgenetic left gonad demonstrated a significantly higher proportion (P = 0.005) of cells carrying a Y chromosome (46.3%) than the streak gonad from the right side (33.9%). Histomorphological examination of the left gonad revealed immature testicular tissue and rete-like structures as well as irregular ovarian type areas with cystic follicular structures. Interphase FISH analysis of the different tissues of this dysgenetic gonad demonstrated variable proportions of cells with an X and a Y chromosome. Whereas Sertoli cells and rete-like structures revealed a significantly higher proportion of XY cells in relation to the whole section of the dysgenetic gonad (P < 0.0001), almost all granulose-like cells carried no Y chromosome. The proportion of XY/X cells in theca-like cells and Leydig cells was similar to that of the whole dysgenetic gonad. In contrast to these findings, spermatogonia exclusively contained an XY constellation.

De Novo Morphogenesis of Seminiferous Tubules From Dissociated Immature Rat Testicular Cells in Xenografts

Testicular development is initiated with the differentiation of Sertoli cells in the embryonic gonad. The aggregation of Sertoli cells is crucial for the generation of testicular cords and thus for the first sign of male gonadal development. To date, functional testicular tissue has not yet been generated in vitro. The objective of this study was to explore the de novo morphogenesis of testicular tissue from isolated postnatal rat testicular cells using a combination of in vitro culture and ectopic xenografting. Immature rat testicular cells were cultured in either a 2-dimensional (laminin-coated coverglass) or a 3-dimensional (extracellular matrix gel) culture system. Whereas testicular cells cultured on laminin showed a slow morphogenetic cascade resulting in cord formation after about 10 days of culture, cells cultured on extracellular matrix gel assembled to a network of cordlike structures within several hours after plating and formed spherical cell aggregates at day 3. Further progression of the morphogenetic cascade was not obtained in either the 2- or the 3-dimensional culture system. In contrast, structures resembling immature testicular tissue were obtained after xenografting of extracellular matrix gel-enclosed spherical testicular cell aggregates. The grafts were vascularized and contained elongated seminiferous tubules. Histologic analysis revealed the presence of a basement membrane, a histologically normal interstitium containing putative Leydig cells, the establishment of tubule lumen, and the integration of few putative spermatogonia into the seminiferous epithelium. We conclude that immature rat testicular cells carry the full potential to generate all somatic components of a testis in xenografts, thus opening fascinating pathways to study testicular organogenesis.

Intersex: Four cases in one family

Intersex presents a unique challenge to modern medicine. Generally, patients with ambiguous genitalia present in the neonatal period. Due to functional failure of the assigned gender role, some patients may be discovered later in life, as in the presented cases. The four patients, all members of the same family and aged 23, 15, 13 and 4 years, presented with ambiguous genitalia. The family history revealed parental consanguinity and one additional case of genital ambiguity. The eldest two patients had developed gynaecomastia during puberty. Examination of the genitalia revealed penoscrotal hypospadias and bifid scrotum in all the four. Gonads were palpable bilaterally in three of the patients, but in the fourth only one gonad was palpable. Buccal smear was negative for sex chromatin in all the cases. On abdominal ultrasound Müllerian structures were found in the two eldest patients. The karyotype in all four cases was determined as 46XY. Gonadal biopsies revealed immature testicular tissue and azoospermia, as sometimes seen in cryptorchidism. We conclude that these cases should be classified as dysgenetic male pseudohermaphrodites, a rare variety of ambiguous genitalia. In agreement with the patients and their parents, they were assigned the male gender and surgery was performed accordingly. We stress the need for active involvement of patient and parents in the process of gender assignment and awareness of hasty decision making.