BackgroundGerm cell transplantation results in fertile recipients and is the only available approach to functionally investigate the spermatogonial stem cell biology in mammals and probably in other vertebrates. In the current study, we describe a novel non-surgical methodology for efficient spermatogonial transplantation into the testes of adult tilapia (O. niloticus), in which endogenous spermatogenesis had been depleted with the cytostatic drug busulfan.Methodology/Principal FindingsUsing two different tilapia strains, the production of fertile spermatozoa with donor characteristics was demonstrated in adult recipient, which also sired progeny with the donor genotype. Also, after cryopreservation tilapia spermatogonial cells were able to differentiate to spermatozoa in the testes of recipient fishes. These findings indicate that injecting germ cells directly into adult testis facilitates and enable fast generation of donor spermatogenesis and offspring compared to previously described methods.ConclusionTherefore, a new suitable methodology for biotechnological investigations in aquaculture was established, with a high potential to improve the production of commercially valuable fish, generate transgenic animals and preserve endangered fish species.

In association with in vitro culture and transplantation, isolation of spermatogonial stem cells (SSCs) is an excellent approach for investigating spermatogonial physiology in vertebrates. However, in fish, the lack of SSC molecular markers represents a great limitation to identify/purify these cells, rendering it difficult to apply several valuable biotechnologies in fish-farming. Herein, we describe potential molecular markers, which served to phenotypically characterize, cultivate and transplant Nile tilapia SSCs. Immunolocalization revealed that Gfra1 is expressed exclusively in single type A undifferentiated spermatogonia (Aund, presumptive SSCs). Likewise, the expression of Nanos2 protein was observed in Aund cells. However, Nanos2-positive spermatogonia have also been identified in cysts with two to eight germ cells that encompass type A differentiated spermatogonia (Adiff). Moreover, we also established effective primary culture conditions that allowed the Nile tilapia spermatogonia to expand their population for at least one month while conserving their original undifferentiated (stemness) characteristics. The maintenance of Aund spermatogonial phenotype was demonstrated by the expression of early germ cell specific markers and, more convincingly, by their ability to colonize and develop in the busulfan-treated adult Nile tilapia recipient testes after germ cell transplantation. In addition to advancing our knowledge on the identity and physiology of fish SSCs, these findings provide the first step in establishing a system that will allow fish SSCs expansion in vitro, representing an important progress towards the development of new biotechnologies in aquaculture, including the possibility of producing transgenic fish.

Summary Testosterone synthesis depends on normal Leydig cell (LC) development, but the mechanisms controlling this development remain unclear. We recently demonstrated that androgen receptor (AR) ablation from a proportion of testicular peritubular myoid cells (PTM‐ARKO) did not affect LC number, but resulted in compensated LC failure. The current study extends these investigations, demonstrating that PTM AR signalling is important for normal development, ultrastructure and function of adult LCs. Notably, mRNAs for LC markers [e.g. steroidogenic factor 1 (Nr5a1), insulin‐like growth factor (Igf‐1) and insulin‐like factor 3 (Insl3)] were significantly reduced in adult PTM‐ARKOs, but not all LCs were similarly affected. Two LC sub‐populations were identified, one apparently ‘normal’ sub‐population that expressed adult LC markers and steroidogenic enzymes as in controls, and another ‘abnormal’ sub‐population that had arrested development and only weakly expressed INSL3, luteinizing hormone receptor, and several steroidogenic enzymes. Furthermore, unlike ‘normal’ LCs in PTM‐ARKOs, the ‘abnormal’ LCs did not involute as expected in response to exogenous testosterone. Differential function of these LC sub‐populations is likely to mean that the ‘normal’ LCs work harder to compensate for the ‘abnormal’ LCs to maintain normal serum testosterone. These findings reveal new paracrine mechanisms underlying adult LC development, which can be further investigated using PTM‐ARKOs.

The urethra is the main place of entry for sexually transmitted pathogens. However, there is little literature on the morphology of the urogenital system, principally the urethra and ducts of the sex accessory glands. The Mongolian gerbil is an insectivorous, herbivorous and monogamous rodent with nocturnal habits; it has been used successfully as a laboratory animal since the 1960s. Therefore, the objective of the present paper was to describe the structure and ultrastructure of the urethra and its relations to the ducts of the accessory sex glands of the Mongolian gerbil ( Meriones unguiculatus ), contributing to the understanding of the reproductive biology of the rodent and aiming to provide data for future experimental studies. Conventional techniques of light and scanning electron microscopy were utilized. The urethra and ducts of the accessory sex glands are similar to those of the albino rat and the mouse.However, there is variation in drainage type among accessory sex glands for the inner urethra. The ducts of the seminal vesicle, the ductus deferens, drain their contents independently into the ampullary duct that opens in the urethra. The ducts of the prostate, coagulating and bulbourethral glands drain their contents independently into the urethra.

The gerbil (Meriones unguiculatus) is a rodent native of the arid regions of Mongolia and China. Because the gerbil can be easily bred in laboratory conditions, this species has been largely used as an experimental model in biomedical research. However, there is still little information concerning the testis structure and function in the gerbil. In this regard, we performed a detailed morpho-functional analysis of the gerbil testis and estimated the spermatogenic cycle length utilizing 3H-thymidine as a marker for germ cell progression during their evolution through the spermatogenic process. The stage frequencies of the XII stages characterized according to the acrosome formation and development were (I-XII) 13.8, 10.1, 8.1, 7.8, 4.0, 11.2, 7.5, 7.1, 5.9, 7.6, 8.1, and 8.9. The mean duration of each seminiferous epithelium cycle was determined to be 10.6 +/- 1.0 days and the total duration of spermatogenesis, based on 4.5 cycles, was approximately 47.5 days. The volume density of tubular and interstitial compartments was approximately 92% and 8%, respectively. Based on the volume occupied by seminiferous tubules in the testis and the tubular diameter, about 9 and 18 m of seminiferous tubules were found per testis and per gram of testis, respectively. Twelve primary spermatocytes were formed from each type A1 spermatogonia. The meiotic index was 2.8, indicating that 30% of cell loss occurs during meiosis. The number of Leydig and Sertoli cells per gram of the testis was 28 million and each Sertoli cell was able to support approximately 13 spermatids. The daily sperm production per gram of testis (spermatogenic efficiency) was 33 million. Taken together, these data indicate that, mainly due to the high seminiferous tubule volume density and Sertoli cell support capacity for germ cells, the gerbil presents high spermatogenic efficiency compared with other mammalian species already investigated. The data obtained in the present study might provide the basis for future research involving the reproductive biology in this species.