Marcelo C. Leal

Histological and Stereological Evaluation of Zebrafish (Danio rerio) Spermatogenesis with an Emphasis on Spermatogonial Generations

Abstract The zebrafish has become an important vertebrate model for basic and biomedical research, including the research field of the biology of reproduction. However, very few morphological and stereological data are available regarding zebrafish testis structure and spermatogenesis. In this careful histomorphometric evaluation of the testis, we studied spermatogonial cells using molecular markers, determined the combined duration of meiotic and spermiogenic phases, and examined the formation of the Sertoli cell barrier (tight junctions). We found at least nine spermatogonial generations and propose a morphology-based nomenclature for spermatogonial generations that is compatible with the one used in higher vertebrates. The number of germ cells per cyst increased dramatically (1 to ∼1360 cells) from undifferentiated spermatogonia type A to early spermatids. The combined duration of meiotic and spermiogenic phases is approximately 6 days, one of the shorter periods among the teleost fish investigated to date. The number of Sertoli cells per cyst increased 9-fold during the maturational cycle of spermatogenic cysts and stabilized in the meiotic phase at a ratio of approximately 100 early spermatids per Sertoli cell (Sertoli cell efficiency). Similarly to mammals, Sertoli cell proliferation ceased in the meiotic phase, coinciding with the formation of tight junctions between Sertoli cells. Hence, the events taking place during puberty in the germinal epithelium of mammals seem to recapitulate the “life history” of each individual spermatogenic cyst in zebrafish.

Zebrafish primary testis tissue culture: An approach to study testis function ex vivo

To develop new tools to study the regulation of testis physiology in teleost fish, a medium-term ex vivo organ culture system was adopted for zebrafish testis tissue. The addition of 100nM 11-ketotestosterone to the system supported complete spermatogenesis, as determined by morphological, molecular and immunohistochemical analyses. Under basal conditions, however, the development of differentiated spermatogonia, spermatocytes, and spermatids was seriously disturbed, probably related to the rapid (within 2 days) down-regulation of the steroidogenic system. Forskolin (0.5microM) stimulated acute androgen release from freshly removed tissue and partially prevented down-regulation of the steroidogenic system. The present ex vivo culture system can serve as a tool to evaluate effects of a wide range of substances on the two main functions of the testis, spermatogenesis and hormone production.

Mlh1 Deficiency in Zebrafish Results in Male Sterility and Aneuploid as Well as Triploid Progeny in Females

In most eukaryotes, recombination of homologous chromosomes during meiosis is necessary for proper chromosome pairing and subsequent segregation. The molecular mechanisms of meiosis are still relatively unknown, but numerous genes are known to be involved, among which are many mismatch repair genes. One of them, mlh1, colocalizes with presumptive sites of crossing over, but its exact action remains unclear. We studied meiotic processes in a knockout line for mlh1 in zebrafish. Male mlh1 mutants are sterile and display an arrest in spermatogenesis at metaphase I, resulting in increased testis weight due to accumulation of prophase I spermatocytes. In contrast, females are fully fertile, but their progeny shows high rates of dysmorphology and mortality within the first days of development. SNP-based chromosome analysis shows that this is caused by aneuploidy, resulting from meiosis I chromosomal missegregation. Surprisingly, the small percentage of progeny that develops normally has a complete triploid genome, consisting of both sets of maternal and one set of paternal chromosomes. As adults, these triploid fish are infertile males with wild-type appearance. The frequency of triploid progeny of mlh1 mutant females is much higher than could be expected for random chromosome segregation. Together, these results show that multiple solutions exist for meiotic crossover/segregation problems.

Oestrogen-induced androgen insufficiency results in a reduction of proliferation and differentiation of spermatogonia in the zebrafish testis

Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, many aspects of the role of androgens in adult teleost spermatogenesis have remained elusive. Since oestrogens inhibit androgen synthesis, we used an oestrogen-induced androgen depletion model to identify androgen-dependent stages during adult zebrafish spermatogenesis. Exposure to 10 nM 17beta-oestradiol (E(2)) in vivo at least halved the mass of differentiating germ cells (from type B spermatogonia to spermatids), while type A spermatogonia accumulated. Studies on the cellular dynamics revealed that a reduction of spermatogonial proliferation together with an inhibition of their differentiation to type B spermatogonia were the basis for the oestrogen-mediated disturbance of spermatogenesis. The capacity of the zebrafish testis to produce 11-ketotestosterone as well as the expression of steroidogenesis-related genes was markedly decreased after in vivo oestrogen exposure. Moreover, the androgen-release response to recombinant zebrafish Lh was lost after oestrogen exposure. We conclude that oestrogen exposure caused a state of androgen insufficiency in adult male zebrafish. Since the downregulation of the steroidogenic system as well as the disturbance of spermatogenesis in testicular explants exposed to E(2) ex vivo was much less severe than after in vivo exposure, the main inhibitory effect appears to be exerted via feedback inhibition of gonadotropin release. This experimental set-up helped to identify spermatogonial proliferation and their differentiation as androgen targets in adult zebrafish spermatogenesis.

The role of angiotensin-(1–7) receptor Mas in spermatogenesis in mice and rats

Evidence regarding the components of the renin–angiotensin (Ang) system suggests that this system plays an important role in male reproduction. However, there are few data available in the literature on the effects of Ang‐(1–7) on the male reproductive system. The present study investigated the effects of the genetic deletion and chronic blockage of Ang‐(1–7) receptor Mas on spermatogenesis and male fertility. The localization of Mas in mouse and rat testes was determined by binding assays and immunofluorescence, whereas the testis structure and spermatogenic process were morphologically and stereologically analysed by light microscopy. Ang‐(1–7) binding and immunofluorescence revealed the presence of Mas in the testes of mice and rats. Although the total numbers of Sertoli and Leydig cells per testis and Leydig cell size were similar in both wild‐type and Mas‐deficient mice, Mas−/– animals exhibited a significant reduction in testis weight and a greater volume of apoptotic cells, giant cells and vacuoles in the seminiferous epithelium. In both mice and rats, an increased number of apoptotic cells were found during meiosis. Due to disturbed spermatogenesis, daily sperm production was markedly reduced in Mas−/– mice. Moreover, chronic infusion of A‐779 [an Ang‐(1–7) antagonist] in rats significantly increased the total number of apoptotic cells and primary spermatocytes in particular stages of spermatogenesis. Taken together, these findings strongly suggest that Ang‐(1–7) receptor Mas plays an important role in the regulation of spermatogenesis.

Characterization of testicular expression of P450 17α-hydroxylase, 17,20-lyase in zebrafish and its perturbation by the pharmaceutical fungicide clotrimazole.

The aim of the present study was to characterize P450 17α-hydroxylase/17,20-lyase (cyp17a1) expression in zebrafish and to assess the effect of the pharmaceutical clotrimazole, a known inhibitor of various cytochrome P450 enzyme activities, on testicular gene and protein expression of this enzyme as well as on the testicular release of 11-ketotestosterone (11-KT), a potent androgen in fish. We first showed that cyp17a1 is predominantly expressed in gonads of zebrafish, notably in male. In vivo, clotrimazole induced a concentration-dependent increase of cyp17a1 gene expression and Cyp17-I protein synthesis in zebrafish testis. Using zebrafish testicular explants, we further showed that clotrimazole did not directly affect cyp17a1 expression but that it did inhibit 11-KT release. These novel data deserve further studies on the effect of azole fungicides on gonadal steroidogenesis.

Sperm cryopreservation affects postthaw motility, but not embryogenesis or larval growth in the Brazilian fish Brycon insignis (Characiformes)

Sperm cryopreservation is an important method for preserving genetic information and facilitating artificial reproduction. The objective was to investigate whether the cryopreservation process affects postthaw sperm motility, embryogenesis, and larval growth in the fish Brycon insignis. Sperm was diluted in methyl glycol and Beltsville Thawing solution, frozen in a nitrogen vapor vessel (dry shipper) and stored in liquid nitrogen. Half of the samples were evaluated both subjectively (% of motile sperm and motility quality score-arbitrary grading system from 0 [no movement] to 5 [rapidly swimming sperm]) and in a computer-assisted sperm analyzer (CASA; percentage of motile sperm and velocity). The other half was used for fertilization and the evaluation of embryogenesis (cleavage and gastrula stages), hatching rate, percentage of larvae with normal development and larval growth up to 112 days posthatching (dph). Fresh sperm was analyzed subjectively (percentage of motile sperm and motility quality score) and used as the control. In the subjective analysis, sperm motility significantly decreased from 100% motile sperm and quality score of 5 in fresh sperm to 54% motile sperm and quality score of 3 after thawing. Under computer-assisted sperm analyzer evaluation, postthaw sperm had 67% motile sperm, 122 μm/sec of curvilinear velocity, 87 μm/sec of straight-line velocity and 103 μm/sec of average path velocity. There were no significant differences between progenies (pooled data) for the percentage of viable embryos in cleavage (62%) or gastrula stages (24%) or in the hatching rate (24%), percentage of normal hatched larvae (93%), larval body weight (39.8 g), or standard length (12.7 cm) at 112 days posthatching. Based on these findings, cryopreserved sperm can be used as a tool to restore the population of endangered species, such as B. insignis, as well as for aquaculture purposes, without any concern regarding quality of the offspring.

Gamete quality of streaked prochilod Prochilodus lineatus (Characiformes) after GnRHa and dopamine antagonist treatment

The efficiency of Ovaprim™ salmon gonadotropin-releasing hormone agonist (GnRHa) and dopamine antagonist on the induction of spawning and spermiation in Prochilodus lineatus in comparison with the commonly used method using pituitary extract (PE) was evaluated. Females received PE at 0.5 + 5.0 mg/kg and Ovaprim™ at 0.05 + 0.45 ml/kg or at 0.125 + 0.375 ml/kg. All males received a first dose of PE at 0.4 mg/kg and then PE at 4.0 mg/kg or Ovaprim™ at 0.25 ml/kg. Oocyte, egg, larvae and sperm quality were evaluated. All females spawned and oocyte, egg and larvae quality were similar between Ovaprim™-treated (both doses) and PE-treated females. Data from females were pooled and the mean values were: 242 g ova weight, 15% ova index, 1209 oocytes/g ova, 284,539 oocytes/female, 183 oocytes/g body weight, 1.18 mm oocyte diameter, 49% fertilization rate, 43% hatching rate and 89% normal larvae. Sperm quality was similar between Ovaprim™-treated and PE-treated males. Data from males were pooled and the mean values of semen were: volume of 3.0 ml, 14.9 × 109 sperm/ml, osmolality of 283 mOsm/kg, pH of 7.4, 71% motile sperm, 217 μm/s curvilinear velocity, 102 μm/s straight-line velocity and 189 μm/s average path velocity. Ovaprim™ treatment can be used for commercial reproduction of P. lineatus, without any loss of gamete quality in comparison with PE treatment.

Sperm dilution ratio affects post-thaw motility rate and velocity of Prochilodus lineatus (Characiformes) sperm.

There is a lack of standardization in sperm cryopreservation of aquatic organisms and, thus, a necessity of more accurate investigations in all steps of this process. In this study, the effects of sperm dilution ratio on post-thaw sperm quality of Prochilodus lineatus were evaluated. Sperm was diluted in a standard freezing medium (glucose and methyl glycol) at four different ratios (sperm to final volume = 1:5, 1:10, 1:50 or 1:100), frozen in a nitrogen vapour vessel at -170°C and then stored in liquid nitrogen vessel at -196°C. Post-thaw motility rate and velocities (curvilinear = VCL; average path = VAP; straight line = VSL) were determined using a Computer-Assisted Sperm Analyzer (CASA) at 10 and 40 s post-activation. The highest motility rates were observed when sperm was frozen at a ratio of 1:5 (76%) and 1:10 (75%). The highest VCL (225 μm/s) and VAP (203 μm/s) were observed at a ratio of 1:10, while VSL was similar among samples frozen at 1:5, 1:10 and 1:50 (97-124 μm/s). When those parameters were evaluated again 30 s later, motility decreased significantly in samples frozen at a ratio of 1:5 (57%) and 1:10 (61%), while velocities decreased significantly in all samples regardless of dilution ratio (75-85 μm/s of VCL, 38-53 μm/s of VAP and 25-39 μm/s of VSL). P. lineatus sperm should be frozen at a ratio of 1:10, where both the number of loaded sperm per straw and the post-thaw quality are maximized.

Storage and transportation of Prochilodus lineatus (Characiformes) sperm prior to cryopreservation

In this study we compared post-thaw quality of P. lineatus sperm frozen shortly after collection, with sperm frozen after dilution and transportation, and up to 6h from collection. From each sperm sample (n=10 males) five aliquots were taken. One aliquot was diluted in the freezing medium (1 sperm:8 glucose:1 methyl glycol) and frozen ∼20min after collection in the field (control), while the other four aliquots were transported to the laboratory where freezing took place 3 or 6h after collection. From the transported aliquots, two were diluted 1:4 in glucose solution before transportation (diluted samples), while the other two were kept undiluted until freezing (undiluted samples). Thus the five treatments were: control, undiluted-3h, diluted-3h, undiluted-6h and diluted-6h. Post-thaw sperm was evaluated for membrane integrity, motility rate and velocities (curvilinear=VCL; average path=VAP; straight line=VSL). Post-thaw membrane integrity did not differ among the five treatments (48-60% intact sperm). Sperm motility rate was similar (P>0.05) between control (64%) and undiluted samples (60-62%) and higher (P<0.05) than that in diluted samples (35-45%), regardless the time after collection when freezing took place. Velocities were higher in control and in undiluted-3h samples (VCL of 254-265μm/s, VAP of 219-244μm/s and VSL of 134-147μm/s) than in diluted samples or samples frozen 6h after collection. P. lineatus sperm can be transported/shipped to the laboratory without decreasing its suitability for cryopreservation. Sperm should be kept undiluted during storage and be frozen within 3h.