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A wild-caught stock of greater amberjack Seriola dumerili reared in sea cages in two commercial aquaculture facilities in Greece was administered gonadotropin-releasing hormone agonist (GnRHa), loaded in controlled release implants, and was used in spawning induction experiments throughout the reproductive season. Sperm quality was evaluated using computer-assisted sperm analysis (CASA) before and after GnRHa administration and the extent of the spermiation period was determined. Males were in spermiation throughout the monitoring period from May 30 until July 18, at temperatures between 19 and 24 °C. However, lower sperm motility duration, density, and survival under cold storage were observed from early July onwards. Sperm quality did not recover after the end of spawning induction experiments in tanks and the return of the fish for 14–28 days to the sea cage in mid-July, which could be related to the high temperatures of this period. An improvement trend was observed in the quality of the milt collected on day 7 after a single GnRHa administration, but a significant decrease was observed on day 21 in sperm density, survival under cold storage, and straight line velocity (VSL). On the contrary, a double GnRHa administration spaced 14 days apart maintained the same sperm quality for a longer period of 29 days. Further spermiation enhancement experiments should be conducted in the future to describe in more detail the kinetics of sperm production after GnRHa therapy and its effects on sperm quality.

Spermatozoa have a streamlined shape to swim through the oviduct to fertilize oocytes. To become svelte spermatozoa, spermatid cytoplasm must be eliminated in several steps including sperm release, which is part of spermiation. Although this process has been well observed, the molecular mechanisms that underlie it remain unclear. In male germ cells, there are membraneless organelles called nuage, which are observed by electron microscopy in various forms of dense material. Reticulated body (RB) and chromatoid body remnant (CR) are two types of nuage in spermatids, but the functions of both are unknown. Using CRISPR/Cas9 technology, we deleted the entire coding sequence of testis-specific serine kinase substrate (TSKS) in mice and demonstrate that TSKS is essential for male fertility through the formation of both RB and CR, prominent sites of TSKS localization. Due to the lack of TSKS-derived nuage (TDN), the cytoplasmic contents cannot be eliminated from spermatid cytoplasm in Tsks knockout mice, resulting in excess residual cytoplasm with an abundance of cytoplasmic materials and inducing an apoptotic response. In addition, ectopic expression of TSKS in cells results in formation of amorphous nuage-like structures; dephosphorylation of TSKS helps to induce nuage, while phosphorylation of TSKS blocks the formation. Our results indicate that TSKS and TDN are essential for spermiation and male fertility by eliminating cytoplasmic contents from the spermatid cytoplasm.

Mammalian spermatogenesis is a highly complex multi-step biological process, and autophagy has been demonstrated to be involved in the process of spermatogenesis. Beclin-1/BECN1, a core autophagy factor, plays a critical role in many biological processes and diseases. However, its function in spermatogenesis remains largely unclear. In the present study, germ cell–specific Beclin 1 (Becn1) knockout mice were generated and were conducted to determine the role of Becn1 in spermatogenesis and fertility of mice. Results indicate that Becn1 deficiency leads to reduced sperm motility and quantity, partial failure of spermiation, actin network disruption, excessive residual cytoplasm, acrosome malformation, and aberrant mitochondrial accumulation of sperm, ultimately resulting in reduced fertility in male mice. Furthermore, inhibition of autophagy was observed in the testes of germ cell–specific Becn1 knockout mice, which may contribute to impaired spermiogenesis and reduced fertility. Collectively, our results reveal that Becn1 is essential for fertility and spermiogenesis in mice. Summary Sentence Autophagy core protein BECN1 is required for male fertility and spermiogenesis through maintaining normal autophagy function in germ cells. Graphical Abstract

Retinoic acid, an active metabolite of vitamin A, is necessary for many developmental processes in mammals. Much of the field of reproduction has looked toward retinoic acid as a key transcriptional regulator and catalyst of differentiation events. This review focuses on the effects of retinoic acid on male and female gamete formation and regulation. Within spermatogenesis, it has been well established that retinoic acid is necessary for the proper formation of the blood–testis barrier, spermatogonial differentiation, spermiation, and assisting in meiotic completion. While many of the roles of retinoic acid in male spermatogenesis are known, investigations into female oogenesis have provided differing results.

Direct evidence for a role of endogenous retinoic acid (RA), the active metabolite of vitamin A in the initial differentiation and meiotic entry of spermatogonia, and thus in the initiation of spermatogenesis is still lacking. RA is synthesized by dedicated enzymes, the retinaldehyde dehydrogenases (RALDH), and binds to and activates nuclear RA receptors (RARA, RARB, and RARG) either within the RA-synthesizing cells or in the neighboring cells. In the present study, we have used a combination of somatic genetic ablations and pharmacological approaches in vivo to show that during the first, prepubertal, spermatogenic cycle (i) RALDH-dependent synthesis of RA by Sertoli cells (SC), the supporting cells of the germ cell (GC) lineage, is indispensable to initiate differentiation of A aligned into A1 spermatogonia; (ii) RARA in SC mediates the effects of RA, possibly through activating Mafb expression, a gene whose Drosophila homolog is mandatory to GC differentiation; (iii) RA synthesized by premeiotic spermatocytes cell autonomously induces meiotic initiation through controlling the RAR-dependent expression of Stra8 . Furthermore, we show that RA of SC origin is no longer necessary for the subsequent spermatogenic cycles but essential to spermiation. Altogether, our data establish that the effects of RA in vivo on spermatogonia differentiation are indirect, via SC, but direct on meiotic initiation in spermatocytes, supporting thereby the notion that, contrary to the situation in the female, RA is necessary to induce meiosis in the male.

Environmental conditions have a significant influence on the physiological processes that regulate the reproduction of fish. However, there is less information on this subject for chondrichthyans, and particularly for males. This study describes the spermatogenic cycle of the narrownose smooth hound shark Mustelus schmitti related to sex steroid plasma levels and the potential influences of photoperiod and temperature as reproductive cues. A total of 70 males were caught bi-monthly for over a year in the coastal waters of Argentina. Testicular samples were taken for histological characterization and blood samples were taken for the measurement of testosterone (T) and progesterone (P 4 ) plasma levels from each male. Bottom temperature was recorded at each sampling point and the daily photoperiod was estimated from Julian Day Calendar. Histological assessment revealed that the complete cycle of spermatogenesis in M. schmitti lasts just over a year, with the onset of the new spermatogenic cycle (spermatocyte formation) overlapping with the end (spermiation and filling of the seminiferous ducts) of the previous cycle. This particular phase was associated with the peak in T plasma levels, which was found to be significantly explained by the increase in photoperiod in spring. The rise in temperature above 15–16 °C at the end of spring was significantly associated with a reduction in T plasma levels. Additionally, an elevation in P 4 plasma levels was observed after the photoperiod increase phase. Males would be ready to mate from mid-spring, following female parturition and just before ovulation/pregnancy.

Sturgeon sperm maturation occurs outside the testes during the transit of testicular spermatozoa (TS) through the kidneys and the Wolffian ducts. A method of in vitro TS maturation in sterlet Acipenser ruthenus was used to investigate the effects of temperature and hormonal stimulation of spermiation on the ability of TS to complete this process. Spermatozoa motility parameters after in vitro maturation of testicular sperm, concentrations of sex steroid hormones and testis morphology were studied in three groups of sterlet: (1) after overwintering in ponds (OW), (2) adapted to spawning temperature (ST), and (3) adapted to spawning temperature with hormonal induction of spermiation (ST-HI). Blood plasma concentrations of testosterone, 11-ketotestosterone and 17,20β-dihydroxy-pregnenolone increased significantly after hormonal induction of spermiation (group ST-HI). In all groups, TS were not motile. After in vitro sperm maturation, motility was up to 60% only in group ST-HI. The data suggest that the ability of TS to be matured in vitro was not related to the environmental temperature, while hormonal stimulation of spermiation during the spawning season was an absolute requirement for optimal in vitro maturation.

Hormonal manipulation in captivity is an essential strategy to enhance sperm quality and fertilization success for endangered species and restocking programs. The main aim of the present study was to investigate sperm quality, seminal fluid metabolites, and fertilization ability of spermatozoa in the hormonally treated Caspian Kutum ( Rutilus frisii ). Fish were treated with sGnRHa in combination with domperidone (Ovaprim) at a dose of 0 (Control), 0.2 mL kg −1 , and 0.4 mL kg −1 of body weight during their natural spawning season. The control group was injected with 0.9% NaCl. Sperm volume (the amount of sperm taken from fish) and sperm quantity (sperm volume kg −1 fish) were higher in hormone-treated groups than non-treated fish ( P  < 0.05). The highest spermatozoa motility, duration of motility, and total amount of produced sperm were observed in hormone-treated groups ( P  < 0.05). Both spermatocrit and spermatozoa concentration were decreased in 0.4 mL kg −1 treatment compared to the control group ( P  < 0.05). The highest calcium concentration in seminal fluid was recorded in 0.4 mL kg −1 treatment that was differed compared to control group ( P  < 0.05). Moreover, the highest seminal fluid osmolality was measured in hormonal-treated fish ( P  < 0.05). In vitro fertilization showed the highest fertilization rate (94.2 ± 2.4%) in fish treated with 0.2 mL kg −1 sGnRHa in combination with domperidone ( P  < 0.05). Also, better hatching rates (87.5–89.8%) were observed in hormonally-induced treatments ( P  < 0.05). The results show the highest reproductive performance in fish treated with 0.2 ml kg −1 sGnRHa in combination with domperidone (Ovaprim) which can be considered to improve the process of controlled reproduction and restocking programs of Caspian Kutum in hatchery centers.

Interspecific hybridisation is common in aquaculture. The maturation of hybrids is an important issue for successive breeding and reducing the risk of genetic pollution if they escape from sea cages. This study examined the maturation of Kue-Tama, the hybrid of the longtooth grouper Epinephelus bruneus  × giant grouper E. lanceolatus (LGGG) produced on 16 April 2014. We used 60 LGGG, five of which were treated with methyltestosterone (MT) and the others were not. We assessed the spermiation response (SpR) of all LGGG from March 2018 to May 2020 and sperm motility from September 2019 to May 2020. We also analysed seasonal fluctuations in plasma sex steroids (estradiol 17β [E2] and 11-ketotestosterone [11-KT]) and gonad histology from April 2018 to February 2019. We confirmed SpR in all MT-treated LGGG and in five out of 55 untreated LGGG. Some LGGG from both groups exhibited SpRs throughout the year. The spermatozoa from both groups showed vigorous motility. SpR in MT-treated LGGG occurred at low 11-KT levels (< 0.1 ng/ml), whereas that in untreated LGGG occurred at high levels (> 273.4 ng/ml). The E2 levels in a few LGGG without SpR increased significantly from September 2018 to February 2019. The April gonads were identified as ovaries without histological confirmation, whereas the July and September gonads were verified histologically. One of the three gonads collected in July 2019 contained vitellogenic oocytes. Our study demonstrated that male LGGG hybrids mature throughout the year and that females can undergo vitellogenesis in the subtropical waters and photoperiod conditions of Amami Islands.

Multidisciplinary approaches to conserve threatened species are required to curb biodiversity loss. Globally, amphibians are facing the most severe declines of any vertebrate class. In response, conservation breeding programs have been established in a growing number of amphibian species as a safeguard against further extinction. One of the main challenges to the long-term success of conservation breeding programs is the maintenance of genetic diversity, which, if lost, poses threats to the viability and adaptive potential of at-risk populations. Integrating reproductive technologies into conservation breeding programs can greatly assist genetic management and facilitate genetic exchange between captive and wild populations, as well as reinvigorate genetic diversity from expired genotypes. The generation of offspring produced via assisted fertilisation using frozen–thawed sperm has been achieved in a small but growing number of amphibian species and is poised to be a valuable tool for the genetic management of many more threatened species globally. This review discusses the role of sperm storage in amphibian conservation, presents the state of current technologies for the short-term cold storage and cryopreservation of amphibian sperm, and discusses the generation of cryo-derived offspring.