Decreased spermatozoal survivability associated with aberrant morphology of the ductuli efferentes proximales of the chicken (Gallus domesticus)

The objective of this research were twofold: 1) to determine if decreased spermatozoal longevity, a previously reported heritable trait in chickens, was attributable to spermatozoal passage through the excurrent ducts, and 2) to document the morphology of the testicular excurrent ducts from affected roosters. Though spermatozoa were viable at ejaculation, as evidenced by their exclusion of ethidium bromide, fertility after intravaginal insemination of spermatozoa from affected roosters was less (p less than 0.001) than that observed with spermatozoa from nonaffected controls, 37 +/- 2.3 versus 58 +/- 1.5%, respectively, over a 21-day egg-collection interval. In contrast, fertility after intramagnal insemination of testicular spermatozoa from affected roosters was equivalent (p greater than 0.05) to that of nonaffected controls, 47 +/- 2.2 versus 41 +/- 3.6%, respectively. After intravaginal insemination, neither type of testicular spermatozoa fertilized oocytes. The ductuli efferentes proximales from affected roosters were characterized by a greater luminal cross-sectional area as well as a diminished height and number of longitudinal epithelial folds (p less than 0.005). It was concluded that heritable decreased spermatozoal longevity in the chicken is not attributable to an inherent spermatozoal defect. Rather, the defect is acquired during passage of spermatozoa through the extragonadal ducts of the rooster.     

Effects of gonadotropin-releasing hormone variants on plasma and testicular androgen levels in intact and hypophysectomized male frogs, Rana esculenta.

The effects of vertebrate gonadotropin-releasing hormone (GnRH) variants on plasma and testicular androgen level in intact and hypophysectomized (PDX) male frogs, Rana esculenta, have been investigated. In intact animals, mammalian (m)-GnRH, m-GnRH analog (buserelin), salmon (s)-GnRH, chicken (c) I-GnRH, cII-GnRH, D-Arg6-cII-GnRH (cII-GnRHA), and lamprey (l)-GnRH (1.5 micrograms and 6 micrograms, total dose given on alternate days for 5 days) were able to enhance androgen production showing that specificity of pituitary responsiveness to GnRH variants appears to be low. Chicken II-GnRH was more effective than s-GnRH in eliciting testicular and circulatory androgen level increase. Moreover, in animals treated with 6 micrograms of cII-GnRH and s-GnRH in combination, androgens decreased as compared with animal treated with cII-GnRH only, suggesting that GnRH receptors bind preferentially the s-GnRH form. In PDX animals, buserelin (1.5 and 6 micrograms), cII-GnRH, and its analog (6 micrograms) were able to increase plasma androgen levels whereas testis androgen concentrations were increased by cII-GnRH (1.5 and 6 micrograms), D-Arg6-cII-GnRHA, and buserelin (6 micrograms). Since androgen production in PDX animals is influenced especially by peptides sharing cII-GnRH structure, it is suggested that a testicular cII-GnRH-like material play a role as local modulator of the gonadal activity in Rana esculenta.     

Interleukin-6 expression during normal maturation of the mouse testis

In this study, we examined the cellular origin and the expression levels of interleukin-6 (IL-6) during normal maturation of mouse testis. The levels of IL-6 (protein and mRNA) were higher in testicular homogenates of sexually immature than mature mice. Immunohistochemical staining of testicular tissues of sexually immature and adult mice show that testicular germ cells, at different stages of differentiation, Leydig cells/interstitial cells and peritubular cells express IL-6. Our results demonstrate, for the first time, overexpression of IL-6 in testicular tissues of immature mice, as compared to mature mice, as well as the expression of IL-6 in germ cells of testicular tissues of adult and sexually immature mice. Thus, our results may indicate the involvement of the endocrine system (gonadotropins and testosterone) in the regulation of IL-6, which is involved in the regulation of testicular development, functions and spermatogenesis.     

Gonadotropin action on androgen synthesis in short-term incubation of explants and dispersed cells of the testis of Xenopus laevis

Testis cells of the toad Xenopus laevis were dissociated with collagenase and the cell suspension was enriched for steroidogenic cells by Percoll gradients. Results suggested that cells should be preincubated during a 6-h period before stimulation with gonadotropin. Our results indicate that a 2-h incubation period with gonadotropin was necessary to obtain a significant response. Furthermore, the cells can be maintained in a functional state longer than mammalian testis cells. Different gonadotropins were used to stimulate androgen production, and their effects were compared in both dissociated cells and testicular explants. Cells were more sensitive to luteinizing hormone (LH) and follicle stimulating hormone (FSH) than the explants (ED50LH = 0.041 +/- 0.003 micrograms for cells and 0.097 +/- 0.002 micrograms for explants: ED50FSH = 0.41 +/- 0.03 micrograms for cells and 0.63 +/- 0.03 micrograms for explants). Moreover, human chorionic gonadotropin (hCG) which only stimulates testicular explants at high doses, failed to stimulate the androgen production of dissociated cells; this indicates a low sensitivity of amphibian testis to hCG and a possible damaging effect of collagenase on the receptors of isolated cells.     

Effect of temperature and the role of testicular descent on post-natal testicular androgen production in the mouse.

Testes from mice aged 3, 15, 25, 30 or 60 days were incubated under basal conditions or in the presence of hCG. One testis from each animal was incubated at 37 degrees C while the contralateral testis was incubated at 32 or 34 degrees C. During development total androgen production in response to hCG (at 32 degrees C) showed a marked increase between 15 and 30 days. The major androgens secreted at this time were testosterone and 5 alpha-androstane-3 alpha,17 beta-diol. There was little change in total androgen production between 30 and 60 days but by 60 days testosterone was the dominant androgen. Both basal and hCG-stimulated androgen production were temperature sensitive. These effects were most pronounced at 30 and 60 days with androgen production significantly inhibited at 37 degrees C. To examine the role of testicular descent in regulating steroidogenesis animals were rendered unilaterally cryptorchid at 19 days of age. At 25 days, when descent is normally completed in the mouse, there was no significant difference in steroidogenesis between scrotal and abdominal testes. By 30 days, however, the steroidogenic potential of the abdominal testis was significantly lower than that of the scrotal testis. These results show that testicular steroidogenesis is sensitive to temperature changes around the time of testicular descent, although descent itself is not required to achieve an adult level of steroidogenesis. The results also show, however, that testicular descent is required to maintain the adult level of steroidogenesis.     

Transferrin overexpression alters testicular function in aged mice

Many studies have shown a correlation between transferrin (Tf) concentration and sperm yield in several mammalian species. We have used transgenic mice expressing human Tf (hTf) to investigate if overexpression of Tf increases the efficiency of mouse spermatogenesis. We demonstrated that a 36% increase of Tf does not ameliorate the efficiency of mouse spermatogenesis but on the contrary resulted in a 36% decrease of testis sperm reserves. Tf overexpression had no effect on testicular determination and development, however testicular function of these transgenic mice was affected in an age-dependent manner. At 16 months of age, testicular and epididymal weights were significantly reduced. While spermatogenesis was qualitatively normal, testicular functions were perturbed. In fact, testosterone rate after human chorionic gonadotropin (hCG) stimulation was lower in Tf overexpressing mice. Intratesticular concentration of estradiol-17beta was increased and fluid accumulation after ligation of rete testis was more abundant in these transgenic mice. Surprisingly, we found that endogenous Tf levels were also increased in Tf overexpressing mice and we demonstrated for the first time that Tf may serve to upregulate its own expression in testis. Collectively, our data show that Tf overexpression has negative effects on testicular function and that Tf levels require strict regulation in the testis.     

Surface of the rooster spermatozoon changes in passing through the Wolffian duct

Fluid secreted by the rooster Wolffian duct contains several proteins separable on polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) gels. Antibodies against these fluid components were obtained by immunizing rabbits, and the IgG fraction was then purified. As judged by indirect immunofluorescence, purified IgG against rooster duct fluid did not bind to any testicular spermatozoa. However, it bound distinctly to the whole surface of spermatozoa from the initial (epididymal) region and more intensely to all spermatozoa from the mid- and terminal regions of the Wolffian duct of the rooster, though not at all to mature duck or pigeon spermatozoa. Thus, in the rooster, as in therian mammals, the surface of the spermatozoon clearly acquires specific components secreted by the Wolffian duct. It should not be assumed that such surface change in rooster spermatozoa is entirely comparable, in a functional sense, to that undergone by mammalian spermatozoa, in which this seems directly related to fertilizing ability. Unlike those of mammals, rooster spermatozoa do not seem to require capacitation, and some spermatozoa in the testis already are competent to fertilize. Components acquired in the Wolffian duct by the rooster spermatozoon may bear on other aspects, perhaps sperm transport and/or survival in the female.     

Sexually dimorphic roles of steroid hormone receptor signaling in gonadal tumorigenesis

Sex steroids control cellular phenotypes by binding to receptor proteins that in turn regulate downstream gene expression. They are important tropic factors in hormonally responsive tissues and have been implicated in the pathogenesis of both benign proliferations and malignancies at some of these sites. Knockout mice lacking inhibins, alpha:beta heterodimeric peptide hormones of the TGFbeta superfamily, develop gonadal tumors that produce sex steroids and depend on pituitary gonadotropin hormones. To better appreciate how sex steroid receptor signaling pathways contribute to the loss of granulosa/Sertoli cell proliferation in the ovary and testis of inhibin alpha (Inhalpha) knockout mice, we are using both pharmacologic and genetic approaches. Roles of androgens in testicular tumor development have been investigated in our previous studies using double-mutant mice lacking inhibins and carrying the null testicular feminization (tfm) mutation of the androgen receptor. Herein, we report that androgens also participate in the development of ovarian tumors, as tumor development is forestalled in mice treated with flutamide, a nonsteroidal inhibitor of androgen actions. Additionally, we generated double-mutant mice lacking estrogen receptor alpha (ERalpha) and Inhalpha or ERbeta and Inhalpha, as well as triple-mutant mice lacking ERalpha, ERbeta, and Inhalpha to determine the effects of individual and combined ER signaling pathways on tumor development. Although estrogens may have proliferative effects during follicle development and are important in specifying the granulosa cell phenotype, ERalpha and ERbeta signaling are not essential for timely granulosa cell tumor development or granulosa cell-like morphological features in ovarian tumors. However, redundant ER signaling through ERalpha and ERbeta in males is critical for testicular tumor formation, as triple-knockout, but not double-knockout, males are protected from early Sertoli cell tumorigenesis and death. Together, these studies indicate important and sexually dimorphic functions of estrogens and androgens in tumor development in this mouse model and indicate, for the first time, overlapping functions of ERalpha and ERbeta in Sertoli cell pathophysiology.     

Gonadotropins, their receptors, and the regulation of testicular functions in fish

The pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate steroidogenesis and spermatogenesis by activating receptors expressed by Leydig cells (LH receptor) and Sertoli cells (FSH receptor), respectively. This concept is also valid in fish, although the piscine receptors may be less discriminatory than their mammalian counterparts. The main biological activity of LH is to regulate Leydig-cell steroid production. Steroidogenesis is moreover modulated in an autoregulatory manner by androgens. The male sex steroids (testosterone in higher vertebrates, 11-ketotestosterone in fish) are required for spermatogenesis, but their mode of action has remained obscure. While piscine FSH also appears to have steroidogenic activity, specific roles have not been described yet in the testis. The feedback of androgens on gonadotrophs presents a complex pattern. Aromatizable androgens/estrogens stimulate LH synthesis in juvenile fish; this effect fades out during maturation. This positive feedback on LH synthesis is balanced by a negative feedback on LH release, which may involve GnRH neurones. While the role of GnRH as LH secretagogue is evident, we have found no indication in adult male African catfish for a direct, GnRH-mediated stimulation of LH synthesis. The limited available information at present precludes a generalized view on the testicular feedback on FSH.     

Analysis of seminal plasma from roosters carrying the Sd (sperm degeneration) allele.

In previous research, subfertile roosters carrying the Sd (sperm degeneration) allele were characterized by malformed proximal efferent ducts. An abnormal biochemical milieu within the excurrent ducts of the testis was inferred. The objective of the present study was to compare seminal plasma composition between mutant (Sd) and normal (sd+) roosters. Phenotypic mutants were selected--on the basis of sperm viability--from a flock of Delaware roosters. After weekly ejaculations, sperm viability was < 60% for mutant roosters as compared to 100% for normal roosters. As reported previously, sperm viability in mutants increased to normal levels after frequent ejaculation. When comparisons were based on semen containing 100% viable spermatozoa, mutants were comparable to normal roosters with respect to sperm concentration and seminal plasma osmolality. Neither genotype was characterized by seminal plasma proteolytic activity at neutral pH. In contrast, seminal plasma from mutants was characterized by an imbalance of electrolytes, amino acids, and protein. Because the rooster lacks accessory sex glands, seminal plasma composition reflects excurrent duct function. Consequently, the abnormal seminal plasma composition of Sd roosters is attributed to excurrent duct dysfunction.     

Development of Fetal Testicular Cells in Androgen Receptor Deficient Mice

During mammalian development, androgens produced by the fetal testis are the most important hormones controlling the masculinization of the reproductive tract and the genitalia. New findings show that the male germ line is the most sensitive to anti-androgenic endocrine disruptors during the embryonic period. In a recent study, we reported that endogenous androgens physiologically control germ cell growth in the male mouse fetus during early fetal life. In the present study, we extended this result by showing the presence of a functional androgen receptor in the gonocytes in the latter part of the fetal life. We also studied the effect of androgens on the development of the somatic testicular cells using the Tfm mice which carry a naturally inactivating mutation of the androgen receptor. Fetal Leydig cell are largely independent of endogenous androgens during fetal development whereas fetal Sertoli cell number is decreased following a default of peritubular myoid cells differenciation. They also point to the gonocyte as a special target for androgens during the embryonic period and indicate a novel mechanism of androgen action on gonocytes. Elucidation of this new pathway in the fetal testis will clarify not only fetal testis physiology but also the effects of environmental anti-androgens that act during fetal life and open new perspectives for future investigations into the sensitivity of fetal germ cell to androgens.     

The cytokine macrophage migration inhibitory factor (MIF) acts as a neurotrophin in the developing inner ear of the zebrafish, Danio rerio

Macrophage migration inhibitory factor (MIF) plays versatile roles in the immune system. MIF is also widely expressed during embryonic development, particularly in the nervous system, although its roles in neural development are only beginning to be understood. Evidence from frogs, mice and zebrafish suggests that MIF has a major role as a neurotrophin in the early development of sensory systems, including the auditory system. Here we show that the zebrafish mif pathway is required for both sensory hair cell (HC) and sensory neuronal cell survival in the ear, for HC differentiation, semicircular canal formation, statoacoustic ganglion (SAG) development, and lateral line HC differentiation. This is consistent with our findings that MIF is expressed in the developing mammalian and avian auditory systems and promotes mouse and chick SAG neurite outgrowth and neuronal survival, demonstrating key instructional roles for MIF in vertebrate otic development.     

Receptor specificity and functional comparison of recombinant sea bass (Dicentrarchus labrax) gonadotropins (FSH and LH) produced in different host systems

Different yields, biopotency, and in vivo pharmacokinetics are obtained for recombinant sea bass gonadoltropins depending on the production system and DNA construct, but they show specific activation of their corresponding receptors. Gonadotropins (GTHs) are glycoprotein hormones that play a major role in the regulation of gonadal functions. Recently, we succeeded in isolating the native sea bass Fsh from sea bass pituitaries, but to ensure the availability of bioactive GTHs and no cross-contamination with other related glycoproteins, recombinant sea bass GTHs were produced using two expression systems-insect and mammalian cells-and different constructs that yielded tethered or noncovalently bound dimers. Their production levels, binding specificity to their homologous cognate receptors, and bioactivity were investigated and compared. Both expression systems were successful in the generation of bioactive recombinant GTHs, but insect Sf9 cells yielded higher amounts of recombinant proteins than mammalian Chinese Hamster Ovary (CHO) stable clones. All recombinant GTHs activated their cognate receptors without cross-ligand binding and were able to stimulate sea bass gonadal steroidogenesis in vitro, although with different biopotencies. To assess their use for in vivo applications, their half-life in sea bass plasma was evaluated. Sf9-GTHs had a lower in vivo stability compared with CHO-GTHs due to their rapid clearance from the blood circulation. Cell-dependent glycosylation could be contributing to the final in vivo stability and biopotency of these recombinant glycoproteins. In conclusion, both insect and mammalian expression systems produced bioactive sea bass recombinant gonadotropins, although with particular features useful for different proposes (e.g., antibody production or in vivo studies, respectively).     

Regulation of testicular steroidogenesis by gonadotropin-releasing hormone agonists and antagonists

Clinical and experimental studies are described on the effects of a gonadotropin-releasing hormone (GnRH) agonist (A) and antagonist (Ant.) on testicular endocrine function. Testicular effects of long-term gonadotropin suppression by GnRH-A were assessed during treatment of prostatic cancer patients. The testis tissue removed after 6 months of A treatment had less than 5% of the testosterone(T)-producing capacity in comparison to testis tissue removed from untreated control patients. However, the LH receptors (R) and responsiveness of T output to LH stimulation in vitro were unchanged. FSH-R decreased by 70%. Hence, despite suppression of gonadotropins and testicular androgen production during long-term GnRH-A treatment the responsiveness to exogenous gonadotropins is maintained. The testicular effects of a gonadotropin suppression induced with GnRH-Ant. and testicular GnRH-R blockade were studied in rats. Besides decreases of gonadotropins and testicular T, systemic Ant. treatment decreased testicular Prl-R, but had no effect on LH-R or FSH-R. Bromocriptine-induced hypoprolactinemia, in contrast, decreased LH-R but had no effect on Prl-R. The results indicate reciprocal regulation of LH-R and Prl-R, and that testicular steroidogenesis and LH-R are under differential regulation, the former by LH, the latter by Prl. In another study, testicular GnRH-R, and consequently the action of a putative testicular GnRH-like factor, were blocked by unilateral intratesticular infusion of Ant. (1 week, Alzet osmotic pumps). The treatment resulted in 90% occupancy of testicular GnRH-R in the Ant.-infused testes, and this was associated with decreased levels of R for LH, FSH and Prl, and of T. The results indicated that the testicular GnRH-R have a physiological function in subtle stimulation of Leydig cell functions.     

Profiling of Androgen Response in Rainbow Trout Pubertal Testis: Relevance to Male Gonad Development and Spermatogenesis

The capacity of testicular somatic cells to promote and sustain germ cell differentiation is largely regulated by sexual steroids and notably androgens. In fish species the importance of androgens is emphasized by their ability to induce sex reversal of the developing fries and to trigger spermatogenesis. Here we studied the influence of androgens on testicular gene expression in trout testis using microarrays. Following treatment of immature males with physiological doses of testosterone or 11-ketotestosterone, 418 genes that exhibit changes in expression were identified. Interestingly, the activity of testosterone appeared stronger than that of 11-ketotestosterone. Expression profiles of responsive genes throughout testis development and in isolated germ cells confirmed androgens to mainly affect gene expression in somatic cells. Furthermore, specific clusters of genes that exhibit regulation coincidently with changes in the natural circulating levels of androgens during the reproductive cycle were highlighted, reinforcing the physiological significance of these data. Among somatic genes, a phylogenetic footprinting study identified putative androgen response elements within the proximal promoter regions of 42 potential direct androgen target genes. Finally, androgens were also found to alter the germ line towards meiotic expression profiles, supporting the hypothesis of a role for the somatic responsive genes in driving germ cell fate. This study significantly increases our understanding of molecular pathways regulated by androgens in vertebrates. The highly cyclic testicular development in trout together with functions associated with regulated genes reveal potential mechanisms for androgen actions in tubule formation, steroid production, germ cell development and sperm secretion.     

Cytoplasmic versus nuclear localization of Fos-related proteins in the frog,Rana esculenta,testis: in vivo and direct in vitro effect of a gonadotropin-releasing hormone agonist

Evidence has been accumulated indicating that GnRH-like peptides are present in a variety of extrabrain areas of mammalian and nonmammalian vertebrates. A pioneer study carried out in the frog, Rana esculenta, demonstrated that testicular GnRH induced spermatogonial proliferation. Recently, we have shown that in proliferating spermatogonia (SPG) of frogs, a change of localization of the oncoprotein Fos, from the cytoplasm to the nucleus, occurs. This leads to the hypothesis that one or more testicular GnRH peptides may regulate SPG proliferation through Fos family proteins. Therefore, in vivo experiments in intact R. esculenta and in vitro incubations of testis fragments have been carried out using GnRH agonist (GnRHa; buserelin) and GnRH antagonist (D-pGlu(1),D-Phe(2),D-Trp(3,6)-GnRH). Cytoplasmic and nuclear Fos-like protein localization has been found by Western blot analysis in testicular extracts. Immunocytochemistry confirmed that cytoplasmic immunostaining was restricted to SPG; change of localization into the nuclear compartment was observed after GnRHa treatment. Northern blot analysis showed that treatments of testis fragments with GnRHa did not modify testicular c-fos mRNA expression. On the contrary, a Fos-like protein of 52 kDa, while not affected in vivo, disappeared from testicular cytosolic extracts after in vitro treatment with GnRHa. Contemporaneously, a 55-kDa Fos-related signal appeared in nuclear extracts. The GnRH antagonist counteracted the effects of GnRHa. Furthermore, in vivo treatments showed that GnRHa acted negatively on a 43-kDa nuclear Fos-related signal and that gonadotropins caused the decrease of 52-kDa cytoplasmic signal. In conclusion, we show, to our knowledge for the first time, that Fos is regulated by GnRHa directly (not through the pituitary) at the testicular level. The main effect appears to be related to Fos translocation from cytoplasmic to nuclear compartments of SPG.     

Influence of gonadotropins on adrenalectomy induced changes in the testis of albino mouse

Effects of adrenalectomy and administration of gonadotropins on cell counts of different cell types of spermatogenesis and morphology of the Leydig cells were studied in 30 day old mice. Adrenalectomy (duration, 12 days; age at autopsy 42 days) caused a significant decrease in the diameters of seminiferous tubules and Leydig cell nucleus and, cell counts of intermediate spermatogonia, round and elongated spermatids. Administration of FSH (75 micrograms/0.1 ml saline) + LH (25 micrograms/0.1 ml saline) everyday for 12 days to adrenalectomized mice restored testicular activity as revealed by significant increases in mean diameter of the Leydig cell nuclei and cell counts of intermediate spermatogonia and elongated spermatids over those of adrenalectomized mice. The results indicate that (i) testis of adrenalectomized mouse responds to gonadotropin treatment and (ii) impairment in gonadotropin secretion is possibly a major factor in inducing testicular regression following adrenalectomy.     

Building a testis: formation of functional testis tissue after transplantation of isolated porcine (Sus scrofa) testis cells

During mammalian development, morphogenesis of the testis requires the coordinated interplay of somatic cells to form seminiferous cords in which the primitive germ cells reside. These cords are the precursor of the functional male gonad and as such form the basis of male fertility. Cell migration during mammalian organogenesis and formation of complex tissues, such as the testis, are difficult to study in situ. Herein, we report extensive rearrangement of cells to regenerate complete functional testis tissue after implantation of isolated neonatal porcine testis cells under the skin of immunodeficient mice. Somatic cells and germ cells reorganized into structures that have remarkable morphologic and physiologic similarity to normal testis tissue, forming the endocrine and spermatogenic compartment of the testis. This unique in vivo system provides an accessible model for the study of testicular morphogenesis that could be especially useful in nonrodent species.