Efferent Ducts Research Articles

The Efferent Ductules in the Lizard Eutropis carinata: a Functional Morphology and Ultrastructural Study

Efferent ductules are part of the male reproductive system. They play a role in providing a nutrient environment for the maintenance of the spermatozoa. Efferent ductules are studied exhaustively in birds and mammals, but reptiles, the first vertebrates that successfully adapted to life on land, have received little attention among the comparative biologists. Hence, the present study aimed to study the seasonal variation in an extra-testicular portion, the efferent ductules, in the male reproductive system of the lizard Eutropis carinata. The results revealed that the epithelium of these ductules is lined by cuboidal non-ciliated cells and ciliated cells. Ultrastructural features of the non-ciliated cells revealed the role of endocytosis and secretion. During the breeding season, the epithelium is positive to periodic Schiff and bromophenol blue staining, indicating the presence of carbohydrate and protein. Histological and histometric parameters exhibit significant seasonal differences. However, there is not much seasonal ultrastructural variation, except for reduced cell height and less cytoplasm as well as reduced granular material in the lumen. Based on the above results, the present study for the first time reveals that the efferent ductules form an essential link between the testis and the epididymis and serve as a conduit for sperm, absorption of rete testis fluid, and secretion. Further, although there is no seasonal variation in the type of cells throughout the length of the efferent ductules during the breeding and non-breeding seasons in E. carinata, the morphology, ultrastructural modifications, activity, and function vary during these seasons.

Calcium, Ca2+-ATPase, Calmodulin, and Calbindin D-28KD Localization in Testis of Leptodactylus chaquensis (Anura: Leptodactylidae).

The intracellular localization of Ca2+, Ca2+-ATPase, Calmodulin, and Calbindin D-28KD have been studied in testes of the toad Leptodactylus chaquensis, using ultracytochemical and immunohistochemical techniques. The Ca2+ presences in the nucleus and into the mitochondria of the germ cells, together with the activity of Ca2+-ATPase detected in the nuclear envelope and mitochondrial crests, suggest the participation of this transporter in the storage of Ca2+. In Sertoli cells, Ca2+ deposits were also found in vesicles and lamellar bodies. Calmodulin and Calbindin D-28KD were revealed in the cytoplasm of both cell types. At the spermatozoon level, the cation deposits were located in the subacrosomal space and in the acrosomal vesicle. Ca2+-ATPase activity was observed in the acrosomal and plasma membranes of the gamete that suggests the existence of a transport system responsible for maintaining low cytoplasmic Ca2+ levels. The activity of Ca2+-ATPase and the location of Ca2+ deposits in gamete tail would be related to flagellar movement. The colocalization of Ca2+ deposits and their binding proteins in efferent duct cells would probably be associated with secretory activity. Considering that intracellular Ca2+ is present in different gonadal cells, this work would provide a better understanding of the cation importance in the testicular functions of this species.

Anatomical Transcriptome Atlas of the Male Mouse Reproductive System During Aging.

The elderly males undergo degenerative fertility and testicular endocrine function that jeopardize the reproductive health and well-being. However, the mechanisms underlying reproductive aging are unclear. Here, we tried to address this by investigating the phenotypes and transcriptomes of seven regions of the male mouse reproductive tract: the testis, efferent ductules, initial segment, caput, corpus and cauda epididymidis, and vas deferens, in adult (3 months) and aged (21 months) mice. Quantitative PCR, immunohistochemistry, immunofluorescent staining, and enzyme-linked immunosorbent assay were performed for the analysis of gene expression in mice, human tissues, and semen samples. Aged male mice showed both systematic and reproductive changes, and remarkable histological changes were detected in the testis and proximal epididymis. Transcriptomes of the male reproductive tract were mapped, and a series of region-specific genes were identified and validated in mouse and/or human tissues, including Protamine 1 (Prm2), ADAM metallopeptidase domain 28 (Adam28), Ribonuclease A family member 13 (Rnase13), WAP four-disulfide core domain 13 (Wfdc13), and Wfdc9. Meanwhile, age-related transcriptome changes of different regions of the male reproductive tract were characterized. Notably, increased immune response was functionally related to the male reproductive aging, especially the T cell activation. An immune response-associated factor, phospholipase A2 group IID (Pla2g2d), was identified as a potential biomarker for reproductive aging in mice. And the PLA2G2D level in human seminal plasma surged at approximately 35 years of age. Furthermore, we highlighted Protein tyrosine phosphatase receptor type C (Ptprc), Lymphocyte protein tyrosine kinase (Lck), Microtubule associated protein tau (Mapt), and Interferon induced protein with tetratricopeptide repeats 3 (Ifit3) as critical molecules in the aging of initial segment, caput, caput, and cauda epididymidis, respectively. This study provides an RNA-seq resource for the male reproductive system during aging in mice, and is expected to improve our understanding of male reproductive aging and infertility.

Identification and selection of healthy spermatozoa in heterozygous carriers of the Phe508del-variant of the CFTR-gene in assisted reproduction

The pathogenic variant Phe508del of the CFTR-gene is the most frequent cause of cystic fibrosis (CF). Whereas male CF-patients are infertile due to bilateral agenesis of the efferent ducts, the fertility status of male heterozygous carriers is uncertain. We aimed at demonstrating the involvement of the CFTR-ion channel during sperm capacitation and to potentially select variant-free spermatozoa in heterozygous carriers of the CFTR-variant using flow cytometry (FC). Using FC and sorting, single cell polymerase chain reaction, immuno-fluorescent staining an experimental study was performed on nine fertile semen donors and three heterozygous infertile men carrying the Phe508del gene variant. Chemical inhibition of CFTR interfered with sperm capacitation. Most viable spermatozoa of heterozygous carriers of the Phe508del variant of the CFTR-gene show immune-fluorescent CFTR. Sperm capacitation in Phe508del carriers was similar to that in healthy semen donors. Distribution of the Phe508del allele in trio data of CF-affected families corresponded to the expected recessive inheritance pattern. Infertility in Phe508del heterozygous men is unlikely to be caused by the pathogenic variant although some contribution cannot be excluded. Normal sperm capacitation in carriers of pathogenic variants of the Phe508del-gene may in part explain the high prevalence of a potentially lethal recessive disorder.

Exosomes released from Sertoli cells contribute to the survival of Leydig cells through CCL20 in rats.

Reciprocal communication between Sertoli and Leydig cells occurs in the testes; however, the detailed mechanisms involved are not completely understood. Exosomes can communicate within neighboring or distant cells to regulate cell function. Our aim was to determine whether exosomes released from Sertoli cells can regulate the survival of Leydig cells. We found that exosomes released from rat primary Sertoli cells could be internalized by Leydig cells in vitro, and promote the survival of Leydig cells, as assessed by optical density at 450 nm, compared to untreated control (mean ± SD: 0.95 ± 0.04 vs 0.79 ± 0.03, P < 0.05). When the exosomes were injected into the interstitial area of rat testis, they could also be internalized by Leydig cells in vivo. To investigate if exosomes released from Sertoli cells can reach Leydig cells in vivo, exosomes were injected into the efferent duct, from where they entered the interstitial space from seminiferous tubules, which indicated that they may cross the blood-testis barrier (BTB). Further in vitro studies found that exosomes released from Sertoli cells significantly increased CC-chemokine ligand 20 (Ccl20) mRNA (mean ± SD: 2.79 ± 0.08 vs 0.98 ± 0.04, P < 0.01) and protein (mean ± SD: 1.08 ± 0.06 vs 0.53 ± 0.05 ng/ml, P < 0.01) levels in Leydig cells, compared to the untreated Leydig cells. CCL20 promoted the phosphorylation of AKT (protein kinase B) in Leydig cells, compared to untreated control (mean ± SD: 0.074 ± 0.002 vs 0.051 ± 0.002, P < 0.01). In conclusion, our results demonstrated that exosomes released by Sertoli cells may cross the BTB and promote the survival of Leydig cells. The findings may add new evidence for Sertoli-Leydig cell communication.

Essential Roles of Efferent Duct Multicilia in Male Fertility.

Cilia are microtubule-based hair-like organelles on the cell surface. Cilia have been implicated in various biological processes ranging from mechanosensation to fluid movement. Ciliary dysfunction leads to a plethora of human diseases, known as ciliopathies. Although non-motile primary cilia are ubiquitous, motile multicilia are found in restricted locations of the body, such as the respiratory tract, the oviduct, the efferent duct, and the brain ventricles. Multicilia beat in a whip-like motion to generate fluid flow over the apical surface of an epithelium. The concerted ciliary motion provides the driving force critical for clearing airway mucus and debris, transporting ova from the ovary to the uterus, maintaining sperm in suspension, and circulating cerebrospinal fluid in the brain. In the male reproductive tract, multiciliated cells (MCCs) were first described in the mid-1800s, but their importance in male fertility remained elusive until recently. MCCs exist in the efferent ducts, which are small, highly convoluted tubules that connect the testis to the epididymis and play an essential role in male fertility. In this review, we will introduce multiciliogenesis, discuss mouse models of male infertility with defective multicilia, and summarize our current knowledge on the biological function of multicilia in the male reproductive tract.

Testicular Atrophy and Epididymitis-Orchitis Associated with Infectious Bronchitis Virus in Broiler Breeder Roosters.

Infectious bronchitis is a respiratory disease of chickens caused by a gammacoronavirus named infectious bronchitis virus (IBV). In addition to affecting the respiratory tract, IBV may also induce urogenital infections, leading to nephropathogenic disease, false layer syndrome in laying hens, and epididymal lithiasis and epididymitis in males. Here, we report a case of decreased reproductive efficiency due to male infertility in 33- to 38-wk-old broiler breeders. At necropsy, the males presented with urates deposited on the skin around the vent and testicular asymmetry due to marked unilateral atrophy. Histopathology revealed lymphocytic epididymitis, epididymal lithiasis, and orchitis. IBV antigen was detected within collecting and efferent ducts of epididymides by immunohistochemistry. IBV strain DMV/1639 was detected by reverse transcription-quantitative PCR in pools of testes, oviducts, tracheas, cecal tonsils, and kidneys from a 37-wk-old affected flock. This report shows evidence of the role of IBV in male chicken infertility and highlights the importance of performing molecular surveillance of IBV to monitor vaccine strains and to detect emerging variants that can potentially hinder production.

Tricresyl phosphate inhibits fertilization in Japanese medaka (Oryzias latipes): Emphasizing metabolic toxicity

As tricresyl phosphate (TCrP) is commonly found in global water sources, its potential reproductive toxicity to fish is of increasing concern. Japanese medaka larvae were exposed to TCrP at 657.9, 1,511, and 4042 ng/L for 100 days. We identified significant fertilization inhibition (6.9%–12.8%) in all exposure groups. Intersex was significantly induced at 4042 ng/L, with an incidence of 22.0%. TCrP exposure also caused dilation of the efferent duct in the testes with maximum duct widths of 83.3, 93.2, and 149.7 μm in the 657.9, 1,511, and 4042 ng/L exposure groups, respectively. These widths were all significantly larger than that observed in the control group (37.7 μm) and likely contributed substantially to fertilization inhibition. The TCrP metabolites 4-OH-MDTP and 3-OH-MDTP, were detected at high concentrations in the liver and elicited 5.8-fold and 5.3-fold greater androgen receptor antagonistic activity than that elicited by TCrP (39.8 μM), which may explain the intersex observed in low exposure groups. 4-OH-MDTP and 3-OH-MDTP elicited anti-estrogenic activities by blocking the estrogen receptor, and the concentrations at which its responses were equal to the IC20 of tamoxifen were 16.1 μM and 18.9 μM, respectively, as detected using the yeast two-hybrid assay. Such anti-estrogenic activities were likely the main driver of dilation of the efferent duct. Observed adverse outcomes after exposure to TCrP all occurred under environmentally relevant concentrations, suggesting considerable ecological risk to wild fish.

Gonadotropin induction of spermiation in Senegalese sole: Effect of temperature and stripping time

Treatments with homologous recombinant follicle-stimulating and luteinizing hormones (rFsh and rLh, respectively) are known to enhance spermatogenesis and sperm production in sole, but the response can be highly variable depending on the dose, duration and time of the year of the rFsh treatment. To further investigate the physiological effects of rFsh and rLh on sperm production in sole, here we examined the pattern of spermiation of F1 males, of approximately 450 g, treated with rFsh and rLh under controlled temperature. In an initial trial at 12 °C, males were weekly injected intramuscularly with 18 μg kg−1 rFsh over five weeks and subsequently treated with a single injection of 18 μg kg−1 rLh. Histological analysis indicated that the rFsh+rLh treatment increased gonad weight and stimulated spermatogenesis, and also enlarged the size of the seminiferous and efferent duct (ED) tubules, resulting in a doubling of sperm production with respect to the controls. Sperm counts in the ED and sequential stripping of males at 24, 48 and 72 h post rLh injection further revealed that only one batch of spermatids is recruited into spermatozoa (Spz) differentiation after a single rLh induction. A peak of sperm accumulation in the ED occurs at 48 h, coinciding with the upregulation of genes potentially involved in Spz maturation. In a second experiment, we tested the effect of two rFsh doses (10 or 18 μg kg−1) over five weeks as previously, followed by one rLh injection at 12 °C or 17 °C. The results confirmed that spermiation was the highest 48 h after rLh treatment at 12 °C, which was increased in a dose-dependent manner with the dose of rFsh previously supplied (from 0.36 to 0.95 × 109 Spz kg−1). However, sperm production elicited with the low rFsh dose was potentiated by ~3-fold (from 0.36 to 1.06 × 109 Spz kg−1) when the rLh treatment was given at 17 °C. These data suggest that in Senegalese sole sperm collection should be carried out at 48 h after rLh treatment, and that a low dose of rFsh at 12 °C is highly efficient for stimulating sperm production when rLh is administered at a temperature close to that occurring during maximum natural spermiation.

Micro-Computed Tomography Reveals a Remarkable Twin Intromittent Organ in Spiders – A Novelty for Arachnids With Direct Sperm Transfer

The modification of male pedipalps into secondary sexual intromittent organs is one of the hallmark characteristics of spiders, yet understanding the development and evolution of male genitalia across the order remains a challenging prospect. The embolus – the sclerite bearing the efferent spermatic duct or spermophor, and used to deliver sperm directly to the female genitalia during copulation – has always been considered the single unambiguously homologous palpal sclerite shared by all spider species, fundamental to the bauplan of the order and to the evolution and functional morphology of spider reproductive systems. Indeed, after two centuries of comparative research on spider reproduction, the presence of a single spermophor and embolus on each of a male spider’s two pedipalps remains a central tenet of evolutionary arachnology. Our findings challenge this premise, and reveal a remarkable twin intromittent organ sperm transfer system in a lineage of Australian palpimanoid spiders, characterized by a bifurcate spermophor and the presence of two efferent ducts leading to a pair of embolic sclerites on each pedipalp. This is the first time such a remarkable conformation has been observed in any group of arachnids with direct sperm transfer, complicating our understanding of palpal sclerite homologies, and challenging ideas about the evolution of spider genitalia.

The Pathology of Infectious Bronchitis.

Infectious bronchitis (IB) is an acute disease of chickens caused by a gammacoronavirus, infectious bronchitis virus (IBV). Infection of the nasal and tracheal mucosa causes a rapid loss of ciliated epithelium and impaired mucociliary clearance that predispose chickens to secondary bacterial infections. In poultry production, disease progression and severity are influenced by other live virus vaccines, immunosuppression, and coexisting pathogens. The digestive tract supports viral replication in the proventriculus, intestines, cloaca, and the bursa of Fabricius. Acute enteritis and stunted growth in young chickens are caused by an enterotropic IBV. IBV spreads systemically by infection of tracheal macrophages and blood monocytes, deep respiratory infections, and potentially ascending viral infection from the cloaca. Nephrotropic IBV causes severe disease in the kidney with necrosis of tubular epithelial cells, inflammation, and renal failure. Viral infection of the female reproductive tract in the first 2 weeks of life causes necrosis and scarring of the oviduct mucosa, resulting in a chronic cystic oviduct that precludes egg formation when the hen matures. Virus infection of mature hens causes necrosis and inflammation of the oviduct mucosa, leading to the deterioration of egg quality and transient interruption of egg production. In males, IBV infection of seminiferous tubules in the testicle and efferent ductules in the epididymis results in epididymitis and epididymal lithiasis, decreases in sperm production and fertility, and viral shed to semen, leading to venereal transmission. The role IBV in gastrointestinal and urogenital disease merits further study.

Histo-morphological features of the efferent ductules of the African straw-coloured fruit bat (Eidolon helium)

Introduction: The African straw-coloured fruit bat plays important roles as pollinators, seed dispersers and important carrier of zoonotic diseases. It is a non-hibernating seasonal breeder whose reproductive biology had received little attention. The aim was to study the histomorphology of the efferent ductules of Eidolon helvum using 10 adult male bats. Methods: The efferent ductules were obtained from the adult bats, fixed by immersion in neutral-buffered formalin and prepared for histology and histochemistry. Results: The result showed that the entire epididymis was enclosed by the thick dense regular connective tissue, tunica albuginea. Within the connective tissue, tubules of efferent ductules occurred in lobules. These lobules were separated from each other by connective tissue trabeculae. The efferent ductules are tiny tubules that connect the rete testis to the initial segments of the epididymis. Each efferent ductule was lined by a simple columnar epithelium of two cell types, containing the ciliated and the non-ciliated cells. The non-ciliated cells were more numerous exhibiting oval shapes, basally located euchromatic nuclei with prominent nucleoli. The ciliated cells were fewer and appeared as dark cells with oval to elongated, centrally or apically located heterochromatic nuclei. Cilia projected from the apical surfaces of the ciliated cells into the lumen. PAS/Alcian blue staining showed presence of PAS positive secretions in the apical cytoplasm of the non-ciliated cells. Significance: These findings indicate that the morphology of efferent ductules of the African straw-coloured fruit bat have similar morphology with most mammals but differ from those of insectivorous bats.

Ontogeny of the testicular excurrent duct system of male Japanese quail (Coturnix japonica): A histological, ultrastructural, and histometric study.

The testicular excurrent duct system undergoes several physiological and morphological changes during the reproductive stage or breeding season in mammals, birds, and reptiles. Studies on normal age-related histomorphological changes in the excurrent duct system of Japanese quails (Coturnix japonica) remain unreported, despite the extensive use of this bird as an avian model in research studies. The current study investigated the histological, ultrastructural, and histometric changes in the testicular excurrent duct system of the Japanese quail during three reproductive stages, namely prepubertal, pubertal, and adult. Simple squamous to low cuboidal cells formed the epithelia of the rete testis in prepubertal and pubertal birds, while in adult birds the lining was low cuboidal to cuboidal. In pubertal and adult birds, the nonciliated Type I epithelial cells of the proximal efferent duct displayed a subapical endocytotic apparatus comprising coated pits, coated apical tubules, and endosomes. There was a significant increase (p ≤ .001) in epithelial heights of all ducts of the excurrent duct system in the mature, sexually active, adult birds when compared to the other age groups. The luminal and tubular diameters, and the cross-sectional areas of efferent ducts and the epididymal duct unit increased significantly (p ≤ .001) with age. It is concluded that the morphology and morphometry of the excurrent ducts of the testis of the Japanese quail change as birds mature.

The Rete Testis: Development and Role in Testis Function

The rete testis connects seminiferous tubules in which germ cells develop to the efferent ducts and the epididymis, where gametes mature and gain mobility. Several recent studies have thoroughly explored the morphogenesis of this structure in mice during embryonic and postnatal periods. A part of the rete testis has been shown to derive from the precursors of gonad somatic cells before sex determination. The other part forms from embryonal Sertoli cells of testis cords adjacent to the mesonephros. The transformation of Sertoli cells into rete testis cells is apparently not limited to the embryonic stage of development and continues during postnatal testis development. Recently, it was found that the rete testis participates in the formation and maintenance of specialized Sertoli cells in terminal segments of seminiferous tubules, transitional zones. Current views suggest that the transitional zones of the seminiferous tubules may represent a niche for spermatogonial stem cells, the site of the prolonged proliferation of Sertoli cells in the pubertal and postpubertal periods of testis development, and also could be a generator of spermatogenic waves. To sum up, the rete testis transports gametes from the testis to the epididymis, maintains pressure within seminiferous tubules, regulates the composition of the testicular fluid, and impacts the spermatogenic process itself.

Bi-allelic mutations in MCIDAS and CCNO cause human infertility associated with abnormal gamete transport.

Reduced generation of multiple motile cilia (RGMC) and the consequent primary ciliary dyskinesia (PCD) cause infertility due to a substantial reduction in the number of multiciliated cells (MCCs) in the efferent ducts (EDs)/oviducts. MCIDAS acts upstream of CCNO to regulate the biogenesis of basal bodies (BBs); therefore, both genes play a vital role in the multiciliogenesis of the reproductive tract epithelium. In this study, whole-exome sequencing was performed to identify the causative genes in 10 unrelated infertile patients with PCD: seven males and three females. Notably, homozygous frameshift mutations in MCIDAS (c.186dupT, p.Pro63Serfs*22) and CCNO (c.262_263insGGCCC, p.Gln88Argfs*8) were identified in one male and one female participant from two unrelated consanguineous families. Haematoxylin-eosin staining/scanning electron microscopy revealed abnormal MCCs in the mutated EDs/oviducts. Furthermore, transmission electron microscopy revealed significantly reduced BBs. Immunofluorescence staining showed the absence of MCIDAS and CCNO signals in the affected tissues and confirmed that MCIDAS acts upstream of CCNO in the context of multiciliogenesis in the reproductive tract epithelium. In vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) was successful, with a positive pregnancy outcome in both MCIDAS- and CCNO-mutated patients. Our results support the use of IVF/ICSI interventions to treat infertility due to RGMC in couples.

Effect of synthetic androgen treatment on the gonad of juvenile longtooth grouper (Epinephelus bruneus)

Commercially significant groupers are protogynous hermaphrodites. Since their sex change from female (ovary) to male (testis) occurred in a larger size and older age, capturing or maintaining males in the aquaculture farm is very difficult. In the present study, juvenile longthooth groupers (2+ years old) were implanted with two doses of 17alpha-methyltestosterone (MT) cholesterol pellet (2 mg or 5 mg MT kg-1 BW) for the induction of artificial sex reversal. Individuals were sampled at 30 and 80 days after treatment. All the control group fish had immature ovaries contained with primary oocytes and germ cells. Fish treated with MT for 30 days had sex-transitional gonad consisted of few primary oocytes and testicular tissues. By using immunohistochemistry, we identified the proliferating and apoptotic cells in the gonad of this sexual stage. After 80 days for MT treatment, gonads of all individuals were filled with germ cells at all stages of spermatogenesis, and the newly formed efferent duct contained active spermatozoa. Since sperm motility was confirmed, MT treatment successfully induced functional sex reversal in pre-pubertal longtooth grouper. This method may dramatically reduce the cost and time required for the production of longtooth grouper.

Examination of testicular lumicrine regulation of activins and immunoregulatory genes in the epididymal caput.

Immunoregulatory genes encoding activin A (Inhba) and B (Inhbb), and indolamine 2,3-dioxygenase-1 (Ido1) are highly expressed in the murine caput epididymidis, which also has a network of intraepithelial mononuclear phagocytes. This environment is postulated to promote immunological tolerance to epididymal sperm. The factors regulating the immunoregulatory agents in the epididymal caput are poorly understood. This study aimed to investigate the potential role of testicular lumicrine factors in regulating activin and other immune-related genes in the caput epididymidis. The efferent ducts in adult C57/Bl6 mice were exposed and ligated bilaterally. Serum and tissues were collected seven days later. Animals with bilateral sham ligation and animals with no ligations (collectively referred to as the "intact" group) were used as controls. Pressure-induced seminiferous epithelial damage due to intratubular fluid accumulation was observed in all ligated testes. Testicular inhibin was significantly increased and testosterone was elevated in some animals following bilateral ligation, but serum testosterone, serum LH, and serum inhibin were normal. Ligation caused epithelial regression in the initial segment, with similar but less severe effects in other caput segments. Activin A staining by immunohistochemistry in the epithelium was reduced in bilateral ligation, particularly in the initial segment, with moderately reduced staining intensity in the rest of the caput. Inhba expression within the caput was not significantly affected by bilateral ligation, but Inhbb was reduced by more than 60%. Transcripts encoding the macrophage-specific receptor Cx3cr1 were significantly reduced following bilateral ligation, but other immune cell markers, Ido1, and inflammatory genes were unaffected. These data indicate that testicular lumicrine secretion regulates several genes that are preferentially expressed in the initial segment, but has marginal effects on genes such as those encoding activin A and IDO1, which are expressed more widely in the caput.

Three-dimensional analysis of mesonephric tubules remodeling into efferent tubules in the male mouse embryo.

Spermatozoa are transported to the epididymal duct through efferent tubules. Although the origin of the efferent tubules is thought to be mesonephric tubules (MTs), their detailed developmental process, for example, where the rete testis and efferent tubules are connected, is unclear. We investigated the structural changes of the MTs in the male mouse embryo using a three-dimensional reconstruction method. Three to six MTs were connected to the Wolffian duct, and some of them branched, resulting in five to nine tips. Rete cells contacted the three to six tips. The MTs showed a folded shape when the gonadal fate was determined. After the testis development started, they became short and straight but emerged as long and twisting by birth. Before birth, the efferent duct was composed of MTs and a cranial portion of the folded Wolffian duct. The contact between the rete testis and efferent tubules is possibly established at the tip of each MT. The MTs regress after gonadal fate is determined but is remodeled to the twisting efferent tubules by birth. The efferent tubules are composed of the MTs but also a cranial portion of the folded Wolffian duct in the mouse.

Gonadal sex differentiation and early ovarian/testicular development in cultured Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel)

Pacific bluefin tuna (PBT), Thunnus orientalis, is one of the most important species for aquaculture in Japan. Recently, the reduction in muscle fat content associated with sexual maturation in farmed PBT has become a serious problem. To develop technologies for inducing sterility, detailed and reliable data on gonadal development in PBT are needed. Here, we demonstrated the process of gonadal sex differentiation, and of early ovarian and testicular development during the immature stages in PBT. Gonadal sex differentiation was first characterized by the formation of the ovarian cavity in female and of the efferent ducts in male 57 days post hatching (dph). The gonads then differentiated into ovaries or testes according to the genotypic sex until 83 dph. During this period, primordial germ cells, oogonia, and type-A spermatogonia were solitarily distributed in the gonads, and the number of germ cells did not differ between sexes. After gonadal sex differentiation, gonads of PBTs developed in a sexually dimorphic manner: proliferation and differentiation of germ cells occurred earlier in the ovaries than in the testes. The oogonia in ovaries formed cysts at 185 dph, but the type-A spermatogonia were solitarily distributed in testes at this stage, and cysts of type-A spermatogonia were first observed at 247 dph. Moreover, the oogonia entered meiosis and differentiated into chromatin-nucleolus stage oocytes until 247 dph, and subsequently into peri-nucleolus stage oocytes until 285 dph, whereas the type-A spermatogonia differentiated into type-B spermatogonia, spermatocytes, spermatids, and spermatozoa from 446 dph onwards. We believe the results of this study provide the necessary basis for future studies on sterile PBT production.

Dynamics of Spermatogenesis and Change in Testicular Morphology under 'Mating' and 'Non-Mating' Conditions in Medaka (Oryzias latipes).

Here, we report that the gross morphology of the testes changes under 'non-mating' or 'mating' conditions in medaka (Oryzias latipes). During these conditions, an efferent duct expands and a histological unit of spermatogenesis, the lobule, increases its number under 'non-mating' conditions. Based on BrdU labeling experiments, lower mitotic activity occurs in gonial cells under 'non-mating' conditions, which is consistent with the reduced number of germ cell cysts. Interestingly, the total number of type A spermatogonia was maintained, regardless of the mating conditions. In addition, the transition from mitosis to meiosis may have been retarded under the 'non-mating' conditions. The minimum time required for germ cells to become sperm, from the onset of commitment to spermatogenesis, was approximately 14 days in vivo. The time was not found to significantly differ between 'non-mating' and 'mating' conditions. The collective data suggest the presence of a mechanism wherein the homeostasis of spermatogenesis is altered in response to the mating conditions.