Host Testes Research Articles

Transient suppression of transplanted spermatogonial stem cell differentiation restores fertility in mice.

SummaryA remarkable feature of tissue stem cells is their ability to regenerate the structure and function of host tissue following transplantation. However, the dynamics of donor stem cells during regeneration remains largely unknown. Here we conducted quantitative clonal fate studies of transplanted mouse spermatogonial stem cells in host seminiferous tubules. We found that, after a large population of donor spermatogonia settle in host testes, through stochastic fate choice, only a small fraction persist and regenerate over the long term, and the rest are lost through differentiation and cell death. Further, based on these insights, we showed how repopulation efficiency can be increased to a level where the fertility of infertile hosts is restored by transiently suppressing differentiation using a chemical inhibitor of retinoic acid synthesis. These findings unlock a range of potential applications of spermatogonial transplantation, from fertility restoration in individuals with cancer to conservation of biological diversity.

Endosialin defines human stem Leydig cells with regenerative potential.

STUDY QUESTIONIs endosialin a specific marker of human stem Leydig cells (SLCs) with the ability to differentiate into testosterone-producing Leydig cells (LCs) in vitro and in vivo?SUMMARY ANSWEREndosialin is a specific marker of human SLCs which differentiate into testosterone-producing LCs in vitro and in vivo.WHAT IS KNOWN ALREADYHuman SLCs have been identified and isolated using the marker platelet-derived growth factor receptor α (PDGFRα) or nerve growth factor receptor (NGFR). However, the specificity was not high; thus, LCs and germ cells could be mistakenly sorted as SLCs if PDGFRα or NGFR was used as a marker for human SLCs isolation.STUDY DESIGN, SIZE, DURATIONFirstly, we re-evaluated the specificity of PDGFRα and NGFR for SLCs in adult human testes. Then we analysed the previously published single-cell sequencing data and found that endosialin may identify human SLCs. Subsequently, we sorted endosialin+ cells from four human donors and characterized their self-renewal and multipotent properties. To assess whether endosialin+ cells have the potential to differentiate into functional LCs in vitro, these cells were stimulated by differentiation-inducing medium. We next assessed the in vivo regenerative potential of human endosialin+ cells after xenotransplantation into the testes of immunodeficient mice.PARTICIPANTS/MATERIALS, SETTING, METHODSSingle-cell sequencing analysis, immunofluorescence and flow cytometry were used to characterize human testis tissues. In vitro colony formation, multipotent differentiation (adipogenic, osteogenic and chondrogenic) and Leydig cell-lineage induction were used to assess stem cell activity. Xenotransplantation into 3-week-old immunodeficient mice was used to determine in vivo regenerative potential. Endpoint measures included testosterone measurements, cell proliferation, immunofluorescence, flow cytometry and quantitative RT–PCR.MAIN RESULTS AND THE ROLE OF CHANCEThe results indicate that endosialin is a specific marker of SLCs compared with PDGFRα and NGFR. Additionally, endosialin+ cells isolated from human testes show extensive proliferation and differentiation potential in vitro: their self-renewal ability was inferred by the formation of spherical clones derived from a single cell. Moreover, these cells could differentiate into functional LCs that secreted testosterone in response to LH in a concentration-dependent manner in vitro. These self-renewal and differentiation properties reinforce the proposal that human testicular endosialin+ cells are SLCs. Furthermore, transplanted human endosialin+ cells appear to colonize the murine host testes, localize to peritubular and perivascular regions, proliferate measurably and differentiate partially into testosterone-producing LCs in vivo.LARGE SCALE DATANA.LIMITATIONS, REASONS FOR CAUTIONOwing to the difficulty in collecting human testis tissue, the sample size was limited. The functions of endosialin on SLCs need to be elucidated in future studies.WIDER IMPLICATIONS OF THE FINDINGSA discriminatory marker, endosialin, for human SLCs purification is a prerequisite to advance research in SLCs and logically promote further clinical translation of SLCs-based therapies for male hypogonadism.STUDY FUNDING/COMPETING INTEREST(S)A.P.X. was supported by the National Key Research and Development Program of China (2017YFA0103802 and 2018YFA0107200). C.D. was supported by the National Natural Science Foundation of China (81971314) and the Natural Science Foundation of Guangdong Province, China (2018B030311039). The authors declare no conflict of interest.

Induction of spermatogenesis under 3-dimentional tissue culture conditions by in vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue

Sperm production is one of the most complex biological processes in the body. In vitro production of sperm is one of the most important goals of researches in the field of male infertility treatment, which is very important in male cancer patients treated with gonadotoxic methods and drugs. In this study, we examine the progression of spermatogenesis after transplantation of spermatogonial stem cells under conditions of testicular tissue culture. Testicular tissue samples from azoospermic patients were obtained and then these were freeze- thawed. Spermatogonial stem cells were isolated by two enzymatic digestion steps and the identification of these cells was confirmed by detecting the PLZF protein. These cells, after being labeled with DiI, were transplanted in azoospermia adult mice model. The host testes were placed on agarose gel as tissue culture system. After 8 weeks, histomorphometric, immunohistochemical and molecular studies were performed. For each experimental group, 3 to 5 NMRI mouse were used at the age of 4 weeks. These mice are treated with Busulfan 40 mg/kg and after 4 weeks, the Azoospermia model is developed. This study is based on 5 biopsy samples taken from different obstructive azoospermic patients. SSCs were isolated by Mirzapour et al (2012) protocol under two steps of enzymatic digestion. SSCs were transplanted into the host testes below the stereo microscope then they were cut into small pieces and placed under 3-D tissue culture conditions on the agarose support layer. The results of histomorphometric studies showed that the mean number of spermatogonial cells, spermatocytes and spermatids in the experimental group was significantly more than the control group (without transplantation) (P<0.05) and most of the cells responded positively to the detection of DiI. Immunohistochemical studies in host testes fragments in the experimental group express the PLZF, SCP3 and ACRBP proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively, which confirmed the human nature of these cells. Also, in molecular studies of PLZF, Tekt1 and TP1, the results indicated that the genes were positive in the test group, while not in the control group. These results suggest that the slow freezing of SSCs can support the induction of spermatogenesis to produce haploid cells under the 3-Dimentional testicular tissue culture.

Transplantation of mouse iPSCs into testis of azoospermic mouse model: in vivo and in vitro study

This study aimed to induce spermatogenesis in azoospermic testis through induced pluripotent stem cells (iPSCs) derived spermatogonial stem cell-like cells (SSCLCs) after iPSCs in vivo and in vitro transplantation and three-dimensional organ culture. DiI-labelled mouse iPSCs were transplanted to azoospermic testis mouse model (pretreated by busulfan 40 mg/kg). This study was designed based on two experimental groups. In experimental group 1(in vivo) labelled iPSCs were transplanted to azoospermic host testis. In experimental group 2 (in vitro) after cell transplantation, fragments of host testes were set as 3D organ culture and testis without cells transplantation served as the control group by the same method. The samples were evaluated by tracing DiI, cell homing, immunohistochemistry, and quantitative RT PCR assays. 2 weeks after iPSCs transplantation, the molecular assessment showed that Plzf, Thy1, Vasa and Gfra1 expression were increased significantly (p ≤ .05) in host testis and labelled iPSCs co-localized by the Plzf and Thy1 markers expression in the base of seminiferous tubules. These findings suggest the ability of iPSCs to achieve homing in the testis niche and indicate the critical inductive role of microenvironment signals in the differentiation of iPSCs to spermatogonial stem cell-like cells.

In vitro transplantation of spermatogonial stem cells isolated from human frozen\u2013thawed testis tissue can induce spermatogenesis under 3-dimensional tissue culture conditions

BackgroundSperm production is one of the most complex biological processes in the body. In vitro production of sperm is one of the most important goals of researches in the field of male infertility treatment, which is very important in male cancer patients treated with gonadotoxic methods and drugs. In this study, we examine the progression of spermatogenesis after transplantation of spermatogonial stem cells under conditions of testicular tissue culture.ResultsTesticular tissue samples from azoospermic patients were obtained and then these were freeze–thawed. Spermatogonial stem cells were isolated by two enzymatic digestion steps and the identification of these cells was confirmed by detecting the PLZF protein. These cells, after being labeled with DiI, were transplanted in azoospermia adult mice model. The host testes were placed on agarose gel as tissue culture system. After 8 weeks, histomorphometric, immunohistochemical and molecular studies were performed. The results of histomorphometric studies showed that the mean number of spermatogonial cells, spermatocytes and spermatids in the experimental group was significantly more than the control group (without transplantation) (P < 0.05) and most of the cells responded positively to the detection of DiI. Immunohistochemical studies in host testes fragments in the experimental group express the PLZF, SCP3 and ACRBP proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively, which confirmed the human nature of these cells. Also, in molecular studies of PLZF, Tekt1 and TP1, the results indicated that the genes were positive in the test group, while not in the control group.ConclusionThese results suggest that the slow freezing of SSCs can support the induction of spermatogenesis to produce haploid cells under the 3-dimensional testicular tissue culture.

TAF4b is required for mouse spermatogonial stem cell development.

Long-term mammalian spermatogenesis requires proper development of spermatogonial stem cells (SSCs) that replenish the testis with germ cell progenitors during adult life. TAF4b is a gonadal-enriched component of the general transcription factor complex, TFIID, which is required for the maintenance of spermatogenesis in the mouse. Successful germ cell transplantation assays into adult TAF4b-deficient host testes suggested that TAF4b performs an essential germ cell autonomous function in SSC establishment and/or maintenance. To elucidate the SSC function of TAF4b, we characterized the initial gonocyte pool and rounds of spermatogenic differentiation in the context of the Taf4b-deficient mouse testis. Here, we demonstrate a significant reduction in the late embryonic gonocyte pool and a deficient expansion of this pool soon after birth. Resulting from this reduction of germ cell progenitors is a developmental delay in meiosis initiation, as compared to age-matched controls. While GFRα1+ spermatogonia are appropriately present as Asingle and Apaired in wild-type testes, TAF4b-deficient testes display an increased proportion of long and clustered chains of GFRα1+ cells. In the absence of TAF4b, seminiferous tubules in the adult testis either lack germ cells altogether or are found to have missing generations of spermatogenic progenitor cells. Together these data indicate that TAF4b-deficient spermatogenic progenitor cells display a tendency for differentiation at the expense of self-renewal and a renewing pool of SSCs fail to establish during the critical window of SSC development.

The relative importance of DNA methylation and Dnmt2-mediated epigenetic regulation on Wolbachia densities and cytoplasmic incompatibility.

Wolbachia pipientis is a worldwide bacterial parasite of arthropods that infects germline cells and manipulates host reproduction to increase the ratio of infected females, the transmitting sex of the bacteria. The most common reproductive manipulation, cytoplasmic incompatibility (CI), is expressed as embryonic death in crosses between infected males and uninfected females. Specifically, Wolbachia modify developing sperm in the testes by unknown means to cause a post-fertilization disruption of the sperm chromatin that incapacitates the first mitosis of the embryo. As these Wolbachia-induced changes are stable, reversible, and affect the host cell cycle machinery including DNA replication and chromosome segregation, we hypothesized that the host methylation pathway is targeted for modulation during cytoplasmic incompatibility because it accounts for all of these traits. Here we show that infection of the testes is associated with a 55% increase of host DNA methylation in Drosophila melanogaster, but methylation of the paternal genome does not correlate with penetrance of CI. Overexpression and knock out of the Drosophila DNA methyltransferase Dnmt2 neither induces nor increases CI. Instead, overexpression decreases Wolbachia titers in host testes by approximately 17%, leading to a similar reduction in CI levels. Finally, strength of CI induced by several different strains of Wolbachia does not correlate with levels of DNA methylation in the host testes. We conclude that DNA methylation mediated by Drosophila’s only known methyltransferase is not required for the transgenerational sperm modification that causes CI.

Elucidating the identity and behavior of spermatogenic stem cells in the mouse testis

Spermatogenesis in mice and other mammalians is supported by a robust stem cell system. Stem cells maintain themselves and continue to produce progeny that will differentiate into sperm over a long period. The pioneering studies conducted from the 1950s to the 1970s, which were based largely on extensive morphological analyses, have established the fundamentals of mammalian spermatogenesis and its stem cells. The prevailing so-called A(single) (A(s)) model, which was originally established in 1971, proposes that singly isolated A(s) spermatogonia are in fact the stem cells. In 1994, the first functional stem cell assay was established based on the formation of repopulating colonies after transplantation in germ cell-depleted host testes, which substantially accelerated the understanding of spermatogenic stem cells. However, because testicular tissues are dissociated into single-cell suspension before transplantation, it was impossible to evaluate the A(s) and other classical models solely by this technique. From 2007 onwards, functional assessment of stem cells without destroying the tissue architecture has become feasible by means of pulse-labeling and live-imaging strategies. Results obtained from these experiments have been challenging the classical thought of stem cells, in which stem cells are a limited number of specialized cells undergoing asymmetric division to produce one self-renewing and one differentiating daughter cells. In contrast, the emerging data suggest that an extended and heterogeneous population of cells exhibiting different degrees of self-renewing and differentiating probabilities forms a reversible, flexible, and stochastic stem cell system as a population. These features may lead to establishment of a more universal principle on stem cells that is shared by other systems.

Transcriptional Regulation of Spermatogonial Progenitor Development and Meiotic Entry.

TAF4b is a component of the general transcription factor complex TFIID that is required for oogenesis and long-term spermatogenesis in the mouse. Male mice that lack TAF4b complete several rounds of early spermatogenesis, but fail to maintain it throughout adult life. In mammals, the ability to maintain spermatogenesis is largely the function of spermatogonial stem cells (SSCs) and their associated niche that provides continuous production of spermatogenic progenitors throughout most of adult life. Successful germ cell transplantation assays into adult TAF4b-deficient host testes previously demonstrated that TAF4b is apparently dispensable for providing essential components of the adult SSC niche. Here, we begin to test whether TAF4b regulates a germ cell-autonomous program of gene transcription required for the development of self-renewing SSCs in early postnatal life required for full spermatogenic maintenance in the adult. To examine the potential disruption of SSC development, we have compared SSC gene expression signatures in the wild type versus the TAF4b-deficient early postnatal testis. This analysis has revealed a reduction in several key components of the SSC transcriptome including Stra8, Lin28, Mvh and Gfra1. To more precisely examine the SSC compartment, we have enriched for SSCs using Thy1-sorting of the early postnatal testis that similarly revealed gene expression signatures consistent with a reduction in SSC renewal. Most importantly, we have revealed a lag in meiotic entry in the TAF4b-deficient testis that may provide a link between timely establishment of the self-renewing SSCs and their subsequent entry into meiosis. A model of how TAF4b may regulate these distinct processes in spermatogenesis will be presented. (platform)

Donor Sertoli cells transplanted into irradiated rat testes stimulate partial recovery of endogenous spermatogenesis

Irradiation of rat testes leads to the failure to support differentiation of the surviving spermatogonia due to damage of the somatic environment. To determine the involvement of Sertoli cells in this somatic damage, we transplanted seminiferous tubule cells from normal immature GFP-transgenic rats into the testes of irradiated rats. The donor Sertoli cells colonized and developed in the host testes. In many seminiferous tubules, the donor Sertoli cells formed abnormal spherical structures in the lumen, but in some tubules they formed a normal-appearing epithelium, but with only isolated spermatogonia, on the basement membrane. When the donor cells were injected into the interstitial region of the testis, they formed tubule-like structures containing Sertoli cells and occasional isolated spermatogonia, both of donor origin. Surprisingly, in host tubules adjacent to these newly formed donor-cell tubules or adjacent to the endogenous tubules with abnormal donor Sertoli-cell structures, endogenous spermatogonia differentiated to the spermatocyte or even to spermatid stages. Around these newly donor cell-formed tubules and the host tubules with abnormal donor Sertoli-cell structures, many cells including macrophages, which perhaps represented chronic inflammation, accumulated in the interstitium. We conclude that the donor Sertoli cells that colonized the seminiferous tubules did not directly support recovery of spermatogenesis. Instead, the colonizing Sertoli cells acted indirectly on the interstitium to stimulate localized differentiation of endogenous spermatogonia.

Parasitic castration of Plutella xylostella larvae induced by polydnaviruses and venom of Cotesia vestalis and Diadegma semiclausum

In the present study, we used gamma-ray to irradiate the female parasitoids to make wasp eggs infertile, resulting in pseudoparasitization, which allowed the analysis of maternal secretions such as polydnaviruses (PDVs) and venom in the absence of larval secretions or teratocytes by the growing parasitoids. We then investigated the spermatogenesis and components of testicular proteins of male Plutella xylostella larvae pseudoparasitized by two endoparasitoids (Cotesia vestalis and Diadegma semiclausum). The results showed that pseudoparasitism by the two endoparasitoids at the early third instar host larvae both induced smaller testes in size than those of nonparasitized host larvae. Both of them caused parasitic castration, and the degree of castration is almost as severe as in naturally parasitized hosts. This suggested that PDVs and venom played a major role in the degeneration of host testes. There are significant differences in the degree of castration induced by the two endoparasitoids, with respect to testicular growth, testicular protein concentrations, and histological changes of germ cells. Cotesia vestalis bracovirus always has a significantly stronger effect on host testicular growth and development than D. semiclausum ichnovirus. SDS-PAGE analysis indicated that synthesis of P 65 and P 67 proteins were clearly inhibited in testes of hosts that were pseudoparasitized by C. vestalis while reduction in synthesis of other proteins was not evident.

Male Gamete Empowerment

The degree of spermatogenesis varies among infertile men from a barely compromised sperm number in the ejaculate to their complete absence-the condition known as azoospermia. The scarcity of gametes often represents a major hindrance to overcoming spermatogenic failure through the use of assisted fertilization techniques. This induced us to attempt replication of the spermatozoon by "male genome cloning." We then investigated whether it is possible to induce human spermatogenesis in host testes by transplanting germ cells obtained from human testicular biopsy specimens into mouse seminiferous tubules. After plating the sorted germ cells on a feeder layer, one putative spermatogonial stem cell colony proliferated up to day 9 of culture. The production of male gametes by ooplasmic somatic cell haploidization has been tested in our laboratory. Attempts to induce neogametogenesis from embryonic stem cells has yielded mouse offspring following fertilization with sperm-like cells.

Heterotopic Transplantation of Testes in Newly Hatched Chickens and Subsequent Production of Offspring via Intramagnal Insemination1

Transplantation of testicular tissue onto the back of immunodeficient nude mice provides a tool to examine testicular development and preserve fertility in mammals. There is no immunodeficient model in birds, but we recently transplanted ovarian tissue between newly hatched chicks from two lines of chickens and produced donor-derived offspring, showing that experimental transplantation is possible in newly hatched chicks. In the present study testicular tissue from newly hatched Barred Plymouth Rock (BPR) chicks was transplanted under the skin of the back, under the skin of the abdomen, or in the abdomen of White Leghorn chicks that had been surgically castrated and immunocompromised. Recipient birds were killed at 10 mo of age. Transplanted tissue was observed in one of five hosts receiving tissue under the skin of the back, two of five hosts receiving tissue under the skin of the abdomen, and three of five chicks with grafts inside the abdominal cavity. In recipients with no regeneration of host testes, testicular transplants grew to the size of normal testes, and histologic analysis showed active spermatogenesis. Subsequent collection of sperm from two successful transplants and surgical insemination of the sperm into the magna of the oviducts of BPR hens resulted in the production of 24 donor-derived chicks. These results demonstrate that the combination of testicular tissue transplantation with intramagnal insemination can produce viable, normal chicks, which could provide a simple approach for the recuperation of live offspring in avian species.

A Genetic Test of the Role of the Maternal Pronucleus in Wolbachia-Induced Cytoplasmic Incompatibility in Drosophila melanogaster

Cytoplasmic incompatibility (CI) is a reproductive sterility found in arthropods that is caused by the endoparasitic bacteria Wolbachia. In CI, host progeny fail to develop during early embryogenesis if Wolbachia-infected males fertilize uninfected females. It is widely accepted that this lethality is caused by some unknown Wolbachia-induced modification of the paternal nuclear material in the host testes. However, the direct means by which this modification leads to early embryonic death are currently unresolved. Results from previous studies suggested that CI lethality occurs as a result of asynchrony in cell cycle timing between the paternal and maternal pronuclei. This hypothesis can be tested experimentally by the prediction that the Wolbachia-modified paternal pronucleus should support androgenetic development (i.e., from the paternal pronucleus only). Using specific mutations in Drosophila melanogaster that produce androgenetic progeny, we demonstrate that the Wolbachia-induced modification inhibits this type of development. This result suggests that CI occurs independently of the maternal pronucleus and argues against pronuclear asynchrony as the primary cause of CI lethality. We propose that CI occurs instead as the result of either a developmentally incompetent paternal pronucleus or asynchrony between the paternal pronucleus and the cell cycle of the egg cytoplasm.

Lentiviral vector transduction of male germ line stem cells in mice

Transgenesis can be achieved in mice by retroviral transduction of male germ line stem cells (GSCs). However, the transduction efficiency by a Moloney murine leukemia virus (MMLV)-based vector is low, probably due to the characteristically slow cell cycle of stem cells. Since lentiviral vectors can transduce non-dividing cells, they have the potential to efficiently transduce GSCs. Here we report that male GSCs of mice can be transduced in vitro by a lentiviral vector and generate complete spermatogenesis when transplanted into infertile host testes. Transduction efficiencies were comparable to those for MMLV transduction using similar experimental conditions. The results suggest that both lentiviral and MMLV vectors could be effective in transducing GSCs of other species. In addition, these and previous studies suggest that transduction of immature donor stem cells transplanted into immature recipient testes will provide the most efficient system for male germ line modification.

Transplantation of germ cells from glial cell line-derived neurotrophic factor-overexpressing mice to host testes depleted of endogenous spermatogenesis by fractionated irradiation.

With a novel method of eliminating spermatogenesis in host animals, male germ cells isolated from mice with targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) were transplanted to evaluate their ability to reproduce the phenotype previously found in the transgenic animals. Successful depletion of endogenous spermatogenesis was achieved using fractionated ionizing irradiation. A dose of 1.5 Gy followed by a dose of 12 Gy after 24 h reduced the percentage of tubule cross-sections displaying endogenous spermatogenesis to approximately 3% and 10% as evidenced by histologic evaluation of testes at 12 and 21 wk, respectively, after irradiation. At this dose, no apparent harmful side effects were noted in the animals. Upon transplantation, GDNF-overexpressing germ cells were found to be able to repopulate the irradiated testes and to form clusters of spermatogonia-like cells resembling those found in the overexpressing donor mice. The cluster cells in transplanted host testes expressed human GDNF, as had been shown previously for clusters in donor animals, and both were strongly positive for the tyrosine kinase receptor Ret. Thus, we devised an efficient method for depleting the seminiferous epithelium of host mice without appreciable adverse effects. In these host mice, GDNF-overexpressing cells reproduced the aberrant phenotype found in the donor transgenic mice.

Recipient preparation is critical for spermatogonial transplantation in the rat

Testis cell transplantation from mice or rats into recipient mouse seminiferous tubules results in donor cell-derived spermatogenesis in nearly all host testes. Normal spermatozoa are produced and, in the most successful mouse transplantations, the donor haplotype is transmitted to progeny of the recipient. However, few studies have been performed in other species. In this report, we demonstrate that rat and mouse testis cells will generate donor cell-derived spermatogenesis in recipient rat seminiferous tubules. Depletion of endogenous spermatogenesis before donor cell transplantation was more difficult in rat than reported for mouse recipients. A protocol employing treatment of neonatal rats with busulfan was most effective in preparing recipients and allowed more than 90% of testes to be colonized by donor cells. Transplantation of mouse testis cells into rat seminiferous tubules was most successful in recipients made cryptorchid and treated with busulfan. In the best experiments, about 55% of rat testes were colonized by mouse cells. Both rat and mouse donor cell-derived spermatogenesis were improved by treatment of rat recipients with leuprolide, a gonadotropin-releasing hormone agonist. The studies indicated that recipient preparation for spermatogonial stem cell transplantation was critical in the rat and differs from the mouse. However, modification of currently used techniques should allow male germ line stem cell transplantation in many species.

Parasitic castration of Pseudaletia separata by Cotesia kariyai and its association with polydnavirus gene expression

Parasitization by the endoparasitoid Cotesia kariyai caused the inhibition of spermatogenesis of Pseudaletia separata. This phenomenon is called parasitic castration. The degree of castration was dependent on the host stage parasitized. Host parasitized on day 1 of the 4th stadium (the time of primary spermatocyte accumulation), had testicular cells with abnormal chromosomes appearing two days after parasitization, and spermiogenesis was completely inhibited. However, when hosts were parasitized on day 0 of the 6th (final) stadium, the degree of castration was less severe, and elongated cells appeared similar to those found in nonparasitized larvae. Results of this study involving injection of C. kariyai polydnavirus (CkPV) and venom suggested that these wasp components caused the appearance of abnormal chromosomes in specific germ cells, which were in mitotic or meiotic prophases. The amount of CkPV gene expression in host testes increased immediately after parasitization and reached a maximum 12 h later. The early-expressed CkPV gene(s) may be related to the parasitic castration phenomenon.

Inhibition of testicular growth and development in Manduca sexta larvae parasitized by the braconid wasp Cotesia congregata

Tobacco hornworm larvae parasitized by the gregarious larval endoparasitoid Cotesia congregata exhibited an inhibition in testicular growth and development, the extent of which was determined by the age and developmental stage of the host at the time of parasitization. The degree of parasitic castration, as assessed by measurements of testicular volume, was correlated with the stadium in which parasitization occurred. A mathematical formula requiring the measurement of testicular length, width and depth was used to calculate testicular volume. The use of the depth parameter revealed a negative correlation between host weight and testicular volume in parasitized larvae. Testicular volumes of fifth instar hosts, which had been parasitized in the first stadium, were significantly smaller than those originally parasitized as fourth or fifth instar larvae and were not correlated with parasitoid load. Effects of natural parasitism were not duplicated by injections of C. congregata polydnavirus and venom, topical treatment with the juvenile hormone analog methoprene, or starvation of nonparasitized larvae. Larvae receiving virus plus venom or methoprene grew larger due to delayed wandering and had larger testes than controls. Deleterious effects on host testes may be due to the effects of nutrient competition between the developing parasitoid progeny and the gonads, combined with the juvenilizing effects believed to be caused by the polydnavirus.

Embryonic castration of the codling moth, Cydia pomonella by an endoparasitoid, Ascogaster quadridentata

Codling moth larvae parasitized by Ascogaster quadridentata are commonly devoid of any internal reproductive organs and exhibit precocious behaviour. It was found that castration could be avoided if host exposure to the parasitoid was delayed until “late” in host embryogenesis. To be effective, the agent causing castration had to be introduced prior to 64h of host embryonic development. If parasitized after 64h, the diminutive host testes underwent a normal maturation sequence. Pseudoparasitized host pupae have external sex characteristics, but no internal sex tissues, indicating a predilection of the castrating agent for mesodermal rather than ectodermal tissue. Host castration was not required for successful parasitoid emergence. The agent causing the castration and its mechanism are unknown. This is the first evidence of host castration occurring during embryogenesis. Speculation is made regarding the benefit of host castration to the parasitoid.