Germ Cell Maturation Research Articles

Genomic and post-genomic leads toward regulation of spermatogenesis

Declining male fertility without sign of any recovery and limited understanding about mechanisms involved in the intra-testicular regulation of spermatogenesis, withholding clinicians from delivering appropriate line of treatment, are serious causes of concern. Several infertile men are not amenable to treatment because hormonal deficiency or physical obstruction is not the underlying cause. A hope has been generated in the post genomic era where we can have information about the testicular genes and proteins which regulate germ cell division, differentiation and maturation in an interactive manner. Expression of some of these genes and proteins may be governed by classical hormones. However, if the genes are defective (naturally or acquired later in life), mere treatment with hormone(s), as is opted presently by clinicians, would not result into production of sperm. High throughput techniques and post genomic endeavors have generated plethora of data for fundamental and clinical andrology. Appropriate analyses and interlinking of these datasets may provide access to very precise information on a myriad of somatic and germ cell specific genes and proteins. Studies of functional genomics involving cell and age specific expression of some of these testicular genes will not only pin point precise role of certain biomolecules in various steps of spermatogenesis but it will also provide strong basis for the diagnosis and treatment of male infertility. In this review, we present some transcriptomic and proteomic information from various testicular somatic and germ cell studies and discuss how a systems biology approach may be brought in to meaningfully utilize the available information.

The Relation between Adult Dark Spermatogonia and Other Parameters of Fertility Potential in Cryptorchid Testes

The fertility potential of boys with cryptorchidism may be related to the number of adult dark spermatogonia per tubular transverse section in testicular biopsies taken at orchiopexy. Placental-like alkaline phosphatase positive gonocytes in testes within year 1 of life indicate preserved ability for germ cell transformation. We related these parameters to the total number of tubular germ cells and other factors associated with fertility potential. The study comprised 89 boys 0.7 to 3 years old (median age 1.8) who underwent bilateral testicular biopsy at bilateral orchiopexy and provided blood samples for gonadotropins and inhibin B. Of 76 boys with adult dark spermatogonia 44 (58%) had a normal mean number of spermatogonia per tubular transverse section compared to 2 of 13 (15%) without adult dark spermatogonia (p <0.05). In the 30 boys with good fertility potential, including a normal mean number of tubular germ cells, and normal gonadotropins and inhibin B, the mean number of adult dark tubular germ cells was 0.081 vs 0.031 in the 38 with low fertility potential, including impaired tubular germ cells and/or low inhibin B but no reactive increase in gonadotropins (p <0.05). In the 21 patients with increased gonadotropins the mean number of adult dark spermatogonia per tubular transverse section was 0.063. Of the 20 boys with normal mean adult dark spermatogonia per tubular transverse section 12 (60%) had good fertility potential, including a normal mean number of tubular germ cells, normal gonadotropins and normal inhibin B, compared to only 18 of 69 (26%) with an impaired mean number of adult dark spermatogonia per tubular transverse section (p <0.05). Of 46 boys with a normal mean number of tubular germ cells 26 (57%) had placental-like alkaline phosphatase positive cells compared to 14 of 43 (33%) with a decreased mean number of tubular germ cells (p <0.05). The number of placental-like alkaline phosphatase positive gonocytes and adult dark spermatogonia per tubular transverse section are important parameters related to the fertility potential of boys with cryptorchid testes.

Electrophoretic and immunocytochemical analysis of Hsp72 and Hsp73 expression in heat-stressed mouse testis and epididymis

ObjectiveOne of the most profound events in stressed cells is the synthesis of a highly conserved family of proteins, the 'heat shock proteins’ (Hsp). The Hsp70 family is the most diverse, and includes constitutive as well as stress-inducible proteins with overlapping or unique functions in different cell compartments. Elucidation of Hsp70 expression during different stages of spermatogenesis and maturation of germ cells is of particular interest due to their high sensitivity to heat treatment. Study designExpression of the main isoforms of the Hsp70 family (constitutive Hsp73 and stress-inducible Hsp72) was determined in normal and heat-stressed mouse testes and epididymis from sexually mature (60-day-old) mice during spermatogenesis and maturation of germ cells. Immunocytochemical analysis and one- and two-dimensional gel electrophoresis were used to separate mouse testicular and epididymal proteins from saline extracts, followed by Western blotting. ResultsUsing a polyclonal anti-Hsp70 antibody that recognizes both isoforms, inducible Hsp72 expression, was demonstrated immunocytochemically only in heat-stressed tissues, while a high level of constitutive Hsp73 isoform expression was found in both normal and heat-stressed mouse male reproductive tissues. Morphological studies have shown that round and elongated spermatids in the testes, as well as all segments of the epididymis, are most sensitive to heat stress. In the epididymis, the reaction was localized in different cell compartments. ConclusionIn heat stress conditions, Hsp73 is mobilized to prevent apoptosis in the testes and epididymis, and assists Hsp72 in the repair of stress-altered protein conformations.

Smads and cell fate: Distinct roles in specification, development, and tumorigenesis in the testis

According to the World Health Organization, a fertile man typically has a sperm count of 15 million per milliliter of semen. This spermatogenic capacity is determined by appropriate specification, proliferation, differentiation, and maturation of somatic and germ cells, events that begin during fetal development and continue throughout adulthood. These processes are orchestrated by the integration of signaling inputs from hormones and growth factors, including those of several transforming growth factor beta (TGFβ) superfamily ligands. This review summarizes current knowledge of the Smad proteins, which serve functions central to fertility by transducing TGFβ superfamily ligand signals in the testis. The importance of regulated Smad expression and differential utilization in signal transduction for fine-tuning cellular responses to ligands is discussed. We evaluate how primary cell culture studies and analyses of genetically modified mice have revealed distinct roles for specific Smads in primordial germ cell lineage specification, in determining the pace of testicular development and in controlling testicular tumorigenesis. This review also addresses the new insights gained from examining heterozygous mice that exhibit intriguing gene-dosage effects, outcomes that provide a new understanding of how TGFβ superfamily ligands influence testis development and function. Finally, we consider the growing understanding that Smads mediate cross-talk with hormones to play a central role in determining male fertility and reproductive health.

Evaluation of the Effects of STZ-Induced Diabetes onIn VitroFertilization and Early Embryogenesis Processes

The aim of this study was to investigate the effects of experimentally induced diabetes on (a) germ cells, (b) in vitro fertilization (IVF) success rate, and (c) gap junction and cell adhesion molecule gene and protein expressions during the early blastocyst period. Germ cells were obtained from healthy and diabetic rats, analyzed for number, motility, and morphology, and used for IVF. After reaching the early blastocyst stage, the expressions of genes encoding gap junction proteins and cell adhesion molecules were analyzed by quantitative RT-PCR. Histomorphologically and immunohistochemically analyses were also performed. Diabetes significantly affected sperm number and motility and the development of oocytes. Gene expressions of β-catenin and connexin family members and protein expressions of E-cadherin and connexin-43 significantly decreased in groups including germ cells isolated from diabetic rats. Connective tissue growth factor expression increased in groups that included sperm cells isolated from diabetic male rats, whereas mucin-1 expression increased in the group that included oocytes isolated from diabetic female rats paired with sperm cells isolated from healthy male rats. In summary, experimentally induced diabetes was found to influence gap junctions, cell adhesion molecules, and associated proteins which all have important roles in germ cell maturation, fertilization, and development.

Hormones and endocrine disruptors in human seminal plasma

Seminal plasma represents a unique environment for maturation, nutrition, and protection of male germ cells from damaging agents. It contains an array of organic as well as inorganic chemicals, encompassing a number of biologically and immunologically active compounds, including hormones. Seminal plasma contains also various pollutants transferred from outer environment known as endocrine disruptors. They interfere with hormones at the receptor level, act as inhibitors of their biosynthesis, and affect hormone regulation.In this minireview, the main groups of hormones detected in seminal plasma are summarized. Seminal gonadal steroids were investigated mostly with aim to use them as biomarkers of impaired spermatogenesis (sperm count, motility, morphology). Concentrations of hormones in the seminal plasma often differ considerably from the blood plasma levels in dependence on their origin. In some instances (dihydrotestosterone, estradiol), their informative value is higher than determination in blood.Out of peptide hormones detected in seminal plasma, peptides of transforming growth factor beta family, especially antimullerian hormone, and oligopeptides related to thyrotropin releasing hormone have the high informative value, while assessment of seminal gonadotropins and prolactin does not bring advantage over determination in blood.Though there is a large body of information about the endocrine disruptors' impact on male reproduction, especially with their potential role in decline of male reproductive functions within the last decades, there are only scarce reports on their presence in seminal plasma. Herein, the main groups of endocrine disruptors found in seminal plasma are reviewed, and the use of their determination for investigation of fertility disorders is discussed.

Development of malignant germ cells - the genvironmental hypothesis

Human germ cell tumors are of interest because of their epidemiology, clinic and patho-biology. Histologically, they are subdivided into various elements, with similarities to embryogenesis. Recent insight triggered development of a higher order division into five types of human germ cell tumors. In the context of male germ cells, only three are relevant; Type I: teratomas and yolk sac tumors of neonates and infants; Type II: seminomas and nonseminomas of (predominantly) adolescents and adults; and Type III: spermatocytic seminomas of the elderly. Various animal models, both occurring spontaneous or induced, are reported, of which their relevance is still a matter of debate. Recent multidisciplinary studies have led to a significant increase in our understanding of the parameters involved in the earliest pathogenetic steps of human germ cells tumors, particularly the seminomas and nonseminomas (Type II). This paper will discuss a number of interesting insights into the normal and aberrant regulation of germ cell development, resulting in the so-called genvironmental hypothesis. This assumes a subtle interaction between environmental- and (epi)genetic parameters, resulting in clinical/phenotypical characteristics. These influence signaling pathways and thereby developmental processes, including gonadal development, germ cell proliferation, maturation and apoptosis. In the case of a disturbed physiology, either due to the germ cell itself, or the micro-environment, embryonic germ cells, during a specific window of sensitization, might be blocked in their maturation, resulting in carcinoma in situ or gonadoblastoma, the precursors of seminomas and nonseminomas. The level of testicularization of the gonad determines the histological composition of the precursor. These insights will allow a better definition of individuals at risk of developing a germ cell malignancy, and allow a better selection of scientific approaches to elucidate the corresponding pathogenesis.

Human amniotic fluid stem cells have a potential to recover ovarian function in mice with chemotherapy-induced sterility

BackgroundHuman amniotic fluid cells (hAFCs) may differentiate into multiple cell lineages and thus have a great potential to become a donor cell source for regenerative medicine. The ability of hAFCs to differentiate into germ cell and oocyte-like cells has been previously documented. Herein we report the potential use of hAFCs to help restore follicles in clinical condition involving premature ovarian failure.ResultsHuman amniotic fluid was obtained via amniocentesis, yielding a subpopulation of cloned hAFCs that was able to form embryoid bodies (EBs) and differentiate into three embryonic germ layers. Moreover, culture of EBs in medium containing human follicular fluid (HFF) or a germ cell maturation factor cocktail (FAC), expressed germ cells markers such as BLIMP1, STELLA, DAZL, VASA, STRA8, SCP3, SCP1, and GDF9. Furthermore, one cell line was grown from clone cells transfected with lentivirus-GFP and displaying morphological characteristics of mesenchymal cells, had the ability to restore ovarian morphology following cell injection into the ovaries of mice sterilized by intraperitoneal injection of cyclophosphamide and busulphan. Restored ovaries displayed many follicle-enclosed oocytes at all stages of development, but no oocytes or follicles were observed in sterilized mice whose ovaries had been injected with medium only (control). Notably, identification of GFP-labeled cells and immunostaining with anti–human antigen-specific antibodies demonstrated that grafted hAFCs survived and differentiated into granulosa cells which directed oocyte maturation. Furthermore, labeling of ovarian tissue for anti-Müllerian hormone expression, a functional marker of folliculogenesis, was strong in hAFCs-transplanted ovaries but inexistent in negative controls.ConclusionThese findings highlight the possibility of using human amniotic fluid-derived stem cells in regenerative medicine, in particular in the area of reproductive health.

Expression and tissue localization of renalase, a novel soluble FAD-dependent protein, in reproductive/steroidogenic systems

Renalase was initially identified in human kidney as a soluble monoamine oxidase. Here we show that renalase is predominantly expressed in reproductive/steroidogenic systems, with particularly substantial expression in oocytes, granulosa, interstitial and luteal cells of ovary, spermatogenic cells of testis, and cortex of adrenal gland, suggesting its function(s) in maturation of germ cells and steroid hormone regulation. Renalase expression increases in testes and ovaries as mice develop and its expression is further enhanced in the ovaries of pregnant mice, indicating an activity of renalase in reproduction. Gonadotropin-releasing hormone (GnRH) antagonist, cetrorelix, repressed renalase expression in mice ovaries and testes, suggesting that steroids regulate renalase expression. Leptin is an effector and modulator of steroid hormones and reproduction. Surprisingly, knockout of leptin causes a dramatic increase of renalase expression in mice testes. Taken together, our results suggest that reproductive/steroidogenic systems are also the sources for renalase secretion and renalase may play a critical role in reproduction and hormone regulation. This provides a novel insight into understanding the function of renalase.

201 IDENTIFICATION OF CANDIDATE X (INACTIVE)-SPECIFIC TRANSCRIPT (XIST) CODING REGION BY HOMOLOGY ANALYSIS OF SEQUENCE AND RELATIVE EXPRESSION ANALYSIS ON FERTILIZED EMBRYO AND PARTHENOTES IN PIG

X (inactive)-specific transcript (XIST) has been known as long noncoding RNA, which regulates X-chromosome inactivation in female mammals to provide dosage equivalence between male and females. Its important roles in early embryo and stem cells development have been suggested in humans and mice. However, its coding region has been unclear in pigs. To determine the coding region of XIST in pigs, we examined candidate XIST coding region using BLAST, PCR, and sequencing techniques. By comparing pig whole genome sequence (Sus scrofa 10.2) with human, murine, and bovine XIST transcript sequence using BLAST, we selected candidate coding regions of XIST in pig. The result showed that XIST is coded on the minus strand of NW_003612825 contig, and its length was nearly 32 kb, which was similar to the length of human and bovine XIST genes. In order to identify the coding region of XIST in this candidate sequence, we designed 24 primer pairs and RT-PCR was performed using porcine embryonic fibroblast cells (PEF) established from porcine male and female lines.The XIST transcripts were expressed in female PEF but not in male PEF. Thus, by designing candidate intron-spanning primers, we confirmed the existence of introns between the first and last exons. The amplicon sequence was analysed and 5 short exons (&lt;400 bp) that have similarity to the XIST coding region of other characterised species were identified. Moreover, to examine the XIST coding strand in pig genome, we conducted strand-specific reverse transcription. With BLAST homology searches, we confirmed that XIST was coded on the negative strand of the contig on the X chromosome. To analyse stage and origin of XIST transcript in the pig, we subsequently performed real-time PCR using cDNA template synthesised from sperm, oocyte, 1, 2, 4, 8, morular, and blastocyst of fertilized embryo and parthenotes, and intron-spanning primer sets were used. The results revealed that XIST was expressed after maternal to zygote transition (4- to 8-cell stage in pig) and not expressed in mature germ cells (sperm and oocyte). Moreover, parthenotes showed a higher expression level compared with fertilized embryos after the 8-cell stage. These results verify the existence of XIST in pig early embryos and may contribute to the comparative analysis of XIST mechanisms in different species. Further study using FISH and methylation analysis may reveal its mechanisms of action. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; No. 2012006276) and the Biogreen 21 program (PJ0081382011) of the Rural Development Administration, Republic of Korea.

A Histological Study of Oogenesis in the Freshwater MusselAnodonta cygnea(Linnaeus, 1758) in Mira Lagoon, Portugal

ABSTRACT Oogenesis of the freshwater mussel Anodonte cygnea was investigated through histological examination over a 12-month period. All specimens were hermaphrodites, having gonads made up of numerous separate female and male acini. Female acini included oogonia with oocytes at different stages of development. Immature oocytes were attached to the acinar wall by a stalk, while mature oocytes were free within the acinus lumen. Several male and female acini were connected to a single germinal duct filled with mature germ cells. A maturity gradation scale was developed, which incorporated the characteristics of the acini, germinal epithelium, lumen and connective tissue. Although oogenesis seems to occur throughout the year, this scale has shown that oogenesis peaks during the summer months.

Spermatogenesis in reciprocal hybrids of Atlantic salmon (Salmo salar L., 1758) and sea trout (Salmo trutta trutta L., 1758) during their freshwater period

Summary Development of gonads and the course of spermatogenesis were studied in 1–2 years old reciprocal hybrids of Atlantic salmon and Sea trout grown. These fish were produced under controlled conditions and raised in restricted natural water courses. Histological analysis (light microscopy) revealed that spermatogenesis in these two types of hybrids was without significant deviations from normal. Early maturing males produced sperm in their first and second years. The percent of maturing individuals and the sexual cycle was similar in the reciprocal hybrids and close to that of the parent species. In the first year of life early maturing males represented 12 and 13% of trout × salmon and salmon × trout hybrids, respectively, while in the second year this percentage raised to 83 and 100%, respectively. During the first year all maturing males of the reciprocal hybrids underwent incomplete maturation, producing a low number of mature germ cells. This feature is also typical in 1 year old fish of the parental species.

Epigenetic Disruption of the PIWI Pathway in Human Spermatogenic Disorders

Epigenetic changes are involved in a wide range of common human diseases. Although DNA methylation defects are known to be associated with male infertility in mice, their impact on human deficiency of sperm production has yet to be determined. We have assessed the global genomic DNA methylation profiles in human infertile male patients with spermatogenic disorders by using the Infinium Human Methylation27 BeadChip. Three populations were studied: conserved spermatogenesis, spermatogenic failure due to germ cell maturation defects, and Sertoli cell-only syndrome samples. A disease-associated DNA methylation profile, characterized by targeting members of the PIWI-associated RNA (piRNA) processing machinery, was obtained. Bisulfite genomic sequencing and pyrosequencing in a large cohort (n = 46) of samples validated the altered DNA methylation patterns observed in piRNA-processing genes. In particular, male infertility was associated with the promoter hypermethylation-associated silencing of PIWIL2 and TDRD1. The downstream effects mediated by the epigenetic inactivation of the PIWI pathway genes were a defective production of piRNAs and a hypomethylation of the LINE-1 repetitive sequence in the affected patients. Overall, our data suggest that DNA methylation, at least that affecting PIWIL2/TDRD1, has a role in the control of gene expression in spermatogenesis and its imbalance contributes to an unsuccessful germ cell development that might explain a group of male infertility disorders.

Concise Review: Spermatogenesis in an Artificial Three-Dimensional System

Culture of spermatogonial stem cells has been performed under a variety of conditions. Most featured two-dimensional systems, with different types of sera, conditioned media, feeder layers, and growth factors. Some have used three-dimensional (3D) matrices produced from gelatin, collagen, or other material. In spite of their increasingly sophisticated composition, however, complete spermatogenesis in vitro has not yet been achieved. In the seminiferous tubules, spermatogenesis occurs in an environment where cells are embedded in a 3D structure with specific niches regulating each stage of germ cell maturation mediated by hormones and paracrine/autocrine factors. We have recently reported achievement of complete in vitro spermatogenesis of mouse testicular germ cells in a 3D culture system featuring a soft agar matrix. This review discusses the advantages of the 3D culture system for studying the spermatogenic process in its entirety. Also discussed are the steps necessary to expand the applicability of the 3D culture system to human germ cell development and determine the functionality of culture-produced spermatozoa for generating offspring.

Autologous Ectopic Grafting of Cryopreserved Testicular Tissue Preserves the Fertility of Prepubescent Monkeys That Receive Sterilizing Cytotoxic Therapy

Boys faced with future sterility as a result of the need of a sterilizing cancer therapy might avoid this fate by engraftment of cryopreserved immature testicular tissue after therapy is completed. Efforts to address this important survivorship issue have been encouraged by reports of the long-term survival and proliferation of human spermatogonia after xenotransplant of cryopreserved immature testicular tissue into immunocompromised murine hosts. However, spermatogenic arrest at the pachytene spermatocyte stage that occurs in this situation has been associated with a failure in sperm production. In this study, we used a prepubescent simian model to address the possibility that testicular tissue engraftment is insufficiently supported in the model to allow suitable maturation of germ cells. Briefly, we carried out autologous orthotopic grafting of cryopreserved testicular tissue from four prepubescent monkeys and one pubescent rhesus monkey after testicular irradiation and castration of the host animal. Five months after implantation of scrotal grafts, we determined that 3% to 7% of the autografts could be recovered with spermatogenesis proceeding through spermatozoa formation in 13% to 17% of the seminiferous tubules formed in the grafts. In contrast, Sertoli cell-only tubules were detected in parallel xenografts transplanted into immunocompromised mice. Our results show that cryopreservation of testicular tissue from prepubescent primates can maintain the fully functional capacity of spermatogonia to produce sperm, but that host conditions are critical for spermatogenic maturation. Furthermore, our results establish an initial perspective on the quantity of cryopreserved material needed to ensure success in preserving fertility through testicular tissue grafts.

Present and Future Prospects of Male Fertility Preservation for Children and Adolescents

Rapid progress in fertility preservation strategies has led to the investigation of ways in which fertile gametes could be generated from cryopreserved immature testicular tissue. Childhood cancer patients remain the major group that can benefit from these techniques. Other potential candidates include patients undergoing gonadectomy and patients with Klinefelter's syndrome and cryptorchid testes. This review aims to present an overview of the current state of knowledge in experimental germ cell transplantation, testicular tissue transplantation, and germ cell culture as fertility preservation methods for males. We included English articles published in PubMed as well as personal files with the focus on studies including human or nonhuman material. Germ cell and testicular tissue transplantation demonstrate clinical options to mature germ cells from immature primate testicular tissue. The most promising approach involves autologous grafting of immature testicular tissue, whereas germ cell maturation in vitro provides the best strategies to overcome problems of cancer contamination in cryopreserved testicular tissue. Three-dimensional and organ culture systems offer the possibility to differentiate immature male germ cells up to the stage of elongated spermatids. Further characterization of early germ cell development in humans is needed to modify these systems for clinical use.

Is matrix metalloproteinase required in postnatal testicular tubules for germ cell maturation?

Background/AimCryptorchidism may cause infertility by failed transformation of neonatal gonocytes into adult dark spermatogonia, the putative stem cells for spermatogenesis. Gonocytes migrate centrifugally to the tubular basement membrane to become adult dark spermatogonia. Regulation of this transformation remains unknown. We aimed to investigate neonatal rodent testis matrix metalloproteinase (MMP) production to see whether MMPs loosen extracellular matrix between Sertoli cells to facilitate gonocyte movement. MethodsSprague-Dawley rat testes (n = 4-6 per group) were collected at embryonic day 19 (E19) and postnatal (P) days P0 to 10 for immunohistochemistry. Immunofluorescent confocal images were captured for presence of membrane type 1 MMP (MT1-MMP), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 2 (TIMP2), mouse VASA homologue, anti-Müllerian hormone, and androgen receptor in tissue sections. Testicular proteins were analyzed by immunoblotting. ResultsMembrane type 1 MMP was strongly present in gonocytes at E19 then decreased, whereas it increased in testicular somatic cells from P0 to P10. Testicular protein levels of MT1-MMP, MMP2, and androgen receptor were constant from E19 to P10. Anti-Müllerian hormone protein sharply decreased after P2, whereas TIMP2 gradually increased from E19 to P10. Gonocytes migrated to basement membrane at P2 to P6. ConclusionMembrane type 1 MMP, MMP2, and TIMP2 were present in testis from E19 to P10 during gonocyte migration and transformation into spermatogenic stem cells. Increased knowledge about germ cell development may aid efforts to improve fertility in cryptorchidism.

Bilateral Undescended Testes Classified According to Preoperative and Postoperative Status of Gonadotropins and Inhibin B in Relation to Testicular Histopathology at Bilateral Orchiopexy in Infant Boys

In recent series of boys with cryptorchidism gonadotropin levels have been higher and serum inhibin B levels have been lower than normal. To some extent the serum values of inhibin B reflect the state of germinative epithelium in cryptorchid testes. We evaluated whether blood samples of gonadotropins and inhibin B as well as histopathology could be used to classify undescended testes. A total of 69 boys (median age 2 years) who underwent surgery for bilateral cryptorchidism had blood samples taken preoperatively and 3 months to 2.1 years postoperatively. Testicular biopsies were performed bilaterally at orchiopexy. The average germ cell number per tubular transverse tubule was measured. Group 1 included 17 patients with increased follicle-stimulating hormone levels. Serum follicle-stimulating hormone and luteinizing hormone decreased significantly after surgery. In 77% of patients (13 of 17) follicle-stimulating hormone levels were normalized. Of these boys 35% (6 of 17) had a low postoperative serum inhibin B. Group 2 consisted of 27 patients with a decreased germ cell number and/or low preoperative inhibin B, but not increased serum follicle-stimulating hormone or luteinizing hormone. There were no significant postoperative changes in follicle-stimulating hormone and luteinizing hormone. Of these boys 22% (6 of 27) had a low serum inhibin B postoperatively. In group 3 there were 25 patients with a normal germ cell number, normal preoperative serum inhibin B and normal gonadotropins. There were no significant changes in luteinizing hormone and follicle-stimulating hormone postoperatively. Only 1 boy in this group had a low postoperative serum inhibin B. Patients with increased gonadotropin levels may have testicular dysgenesis and some may benefit from early surgery. Patients with normal gonadotropin levels and a decreased germ cell number have transient hypothalamus-pituitary-gonadal hypofunction and a poor fertility prognosis. These patients may benefit from gonadotropin treatment after orchiopexy. Patients with normal gonadotropins, inhibin B and germ cell number have a good fertility prognosis after surgery.

Recovery of Spermatogenesis after Irradiation or Chemotherapy.

Cytotoxic damage to the testis by radiation and chemotherapy primarily kills the most sensitive cells, the rapidly proliferating differentiating spermatogonia, but stem spermatogonia are also damaged and undergo either immediate or delayed cell loss. The surviving stem cells then change from their steady-state self-renewal pattern to enhance recovery processes, likely in response to altered signaling from the somatic environment constituting the niche. At this point the stem cells increase proliferation and self-renewal and their differentiation and cell loss processes are altered. Although the kinetics of recovery varies greatly between mice, rats, macaques, and humans, it is gradual in all species. Recovery kinetics in a given species often seem to occur in parallel after irradiation or treatment with gonadotoxic chemotherapy drugs. Three aspects of the recovery process need to be considered for tissue homeostasis; (1) the recovery of surviving stem cell numbers, (2) the initiation of their differentiation process, and (3) their ability to produce mature germ cells including the spermatozoa. In mice and rats the stem spermatogonia initiate self-renewal within 2 weeks after cytotoxic injury and their number gradually recovers to a maximum level over a 5-month period. In contrast in monkeys and humans, after irradiation there is a progressive decline in the numbers of putative stem cells, the type A spermatogonia, over a period of 2-4 months, then followed by subsequent increases in numbers. Complete recovery of stem cell numbers may be possible after low doses of cytotoxicants but not after higher doses. The species markedly differ with respect to the reinitiation of spermatogonial differentiation. In mice, differentiating cells are observed as soon as 1 week after cytotoxic treatment, but in rats this appears to be more delayed, and in some strains there is an absolute and permanent block in spermatogonial differentiation even at modest doses radiation. In humans, but not monkeys, there is a transient suppression of spermatogonial differentiation, lasting about 4 months, but then it proceeds. Finally in rodents and monkeys where quantitative and histological studies have been performed, higher doses of cytotoxic injury result in a decreased ability of the tissue to support complete differentiation of developing germ cells to post-meiotic stages and result in a permanent decrease in sperm counts. In humans however, many patients, who do recover sperm counts after cancer therapy, recover to their pretreatment range but occasional patients remain severely oligospermic. Failure of recovery appears to be due at least in part to damage to the somatic environment, as we have demonstrated with reciprocal transplantation experiments that irradiated rat testes were not capable of supporting differentiation of normal spermatogonia but the spermatogonia from these irradiated rats normally colonized and differentiated in nude mouse testes. This highlights the importance of assessing somatically derived factors known to influence the self-renewal of spermatogonial stem cells (e.g. GDNF, FGF2, CSF1, IGF-I) and the differentiation of spermatogonia (Neuregulin 1, BMP4, activin A, retinoids) in these models attempting recovery of stem cell number and maintaining spermatogenic cell differentiation to achieve homeostasis of the seminiferous epithelium after cytotoxic injury. (Research supported by NIH ES008075 to MLM and HD061301to GS)

Biological Characteristics of Fish Germ Cells and their Application to Developmental Biotechnology

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.