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Studies in teleosts suggest that progestins have crucial functions during early spermatogenesis. However, the role of the different progestin receptors in these mechanisms is poorly understood. In this work, we investigated the expression pattern and hormonal regulation of the classical nuclear progestin receptor (Pgr) in the gilthead seabream at three different stages of spermatogenesis: the resting (postspawning) phase, onset of spermatogenesis, and spermiation. Immunolocalization experiments using a seabream specific Pgr antibody revealed that the receptor was expressed in Sertoli and Leydig cells, and also in a subset of spermatogonia type A, throughout spermatogenesis. Short-term treatment of testis explants with 17β-estradiol (E2) increased pgr mRNA expression at all stages, while the progestin 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) had the opposite effect. At the resting stage, Sertoli cell Pgr expression was positively correlated with the occurrence of proliferating spermatogonia type A in the tubules, and both processes were incremented in vitro by E2 likely through the estrogen receptor alpha (Era) expressed in Sertoli and Leydig cells. In contrast, treatment with 17,20βP downregulated Pgr expression in somatic cells. The androgen 11-ketotestosterone (11-KT) upregulated pgr expression in Leydig cells and promoted the proliferation of mostly spermatogonia type B, but only during spermiation. No relationship between the changes in the cell type-specific expression of the Pgr with the entry into meiosis of germ cells was found. These data suggest a differential steroid regulation of Pgr expression during seabream spermatogenesis and the potential interplay of the E2/Era and 17,20βP/Pgr pathways for the maintenance of spermatogonial renewal rather than entry into meiosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The mediation of fluid homeostasis by multiple classes of aquaporins has been suggested to be essential during spermatogenesis and spermiation. In the marine teleost gilthead seabream (Sparus aurata), seven distinct aquaporins, Aqp0a, -1aa, -1ab, -7, -8b, -9b and -10b, are differentially expressed in the somatic and germ cell lineages of the spermiating testis, but the endocrine regulation of these channels during germ cell development is unknown. In this study, we investigated the in vivo developmental expression of aquaporins in the seabream testis together with plasma androgen concentrations. We then examined the in vitro regulatory effects of recombinant piscine gonadotropins, follicle-stimulating (rFsh) and luteinizing (rLh) hormones, and sex steroids on aquaporin mRNA levels during the spermatogenic cycle. During the resting phase, when plasma levels of androgens were low, the testis exclusively contained proliferating spermatogonia expressing Aqp1ab, whereas Aqp10b and -9b were localized in Sertoli and Leydig cells, respectively. At the onset of spermatogenesis and during spermiation, the increase of androgen plasma levels correlated with the additional appearance of Aqp0a and -7 in Sertoli cells, Aqp0a in spermatogonia and spermatocytes, Aqp1ab, -7 and -10b from spermatogonia to spermatozoa, and Aqp1aa and -8b in spermatids and spermatozoa. Short-term in vitro incubation of testis explants indicated that most aquaporins in Sertoli cells and early germ cells were upregulated by rFsh and/or rLh through androgen-dependent pathways, although Aqp1ab in proliferating spermatogonia was also activated by estrogens. However, expression of Aqp9b in Leydig cells, and of Aqp1aa and -7 in spermatocytes and spermatids, was also directly stimulated by rLh. These results reveal a complex gonadotropic control of aquaporin expression during seabream germ cell development, apparently involving both androgen-dependent and independent pathways, which may assure the fine tuning of aquaporin-mediated fluid secretion and absorption mechanisms in the seabream testis.

Fluid homeostasis is recognized as a critical factor during the development, maturation, and function of vertebrate male germ cells. These processes have been associated with the presence of multiple members of the aquaporin superfamily of water and solute channels in different cell types along the reproductive tract as well as in spermatozoa. We present a comparative analysis of the existing knowledge of aquaporin biology in the male reproductive tissues of mammals and teleosts. Current data suggest that in both vertebrate groups, aquaporins may have similar functions during differentiation of spermatozoa in the germinal epithelium, in the concentration and maturation of sperm in the testicular ducts, and in the regulation of osmotically induced volume changes in ejaculated spermatozoa. Recent studies have also provided insight into the possible function of aquaporins beyond water transport, such as in signaling pathways during spermatogenesis or the sensing of cell swelling and mitochondrial peroxide transport in activated sperm. However, an understanding of the specific physiological functions of the various aquaporins during germ cell development and sperm motility, as well as the molecular mechanisms involved, remains elusive. Novel experimental approaches need to be developed to elucidate these processes and to dissect the regulatory intracellular pathways implicated, which will greatly help to uncover the molecular basis of sperm physiology and male fertility in vertebrates., (© 2015 Marine Biological Laboratory.)

The current view of the control of spermatogenesis by Fsh and Lh in non-mammalian vertebrates is largely based on studies carried out in teleosts with cystic and cyclic spermatogenesis. Much less is known concerning the specific actions of gonadotropins during semicystic germ cell development, a type of spermatogenesis in which germ cells are released into the tubular lumen where they transform into spermatozoa. In this study, using homologous gonadotropins and a candidate gene approach, for which the genes' testicular cell-type-specific expression was established, we investigated the regulatory effects of Fsh and Lh on gene expression during spermatogenesis in Senegalese sole (Solea senegalensis), a flatfish with asynchronous and semicystic germ cell development. During early spermatogenesis, Fsh and Lh upregulated steroidogenesis-related genes and nuclear steroid receptors, expressed in both somatic and germ cells, through steroid-dependent pathways, although Lh preferentially stimulated the expression of downstream genes involved in androgen and progestin syntheses. In addition, Lh specifically promoted the expression of spermatid-specific genes encoding spermatozoan flagellar proteins through direct interaction with the Lh receptor in these cells. Interestingly, at this spermatogenic stage, Fsh primarily regulated genes encoding Sertoli cell growth factors with potentially antagonistic effects on germ cell proliferation and differentiation through steroid mediation. During late spermatogenesis, fewer genes were regulated by Fsh or Lh, which was associated with a translational and posttranslational downregulation of the Fsh receptor in different testicular compartments. These results reveal that conserved and specialized gonadotropic pathways regulate semicystic spermatogenesis in flatfish, which may spatially adjust cell germ development to maintain a continuous reservoir of spermatids in the testis., (© 2014 Society for Endocrinology.)

In both mammals and teleosts, the differentiation of postmeiotic spermatids to spermatozoa (spermiogenesis) is thought to be indirectly controlled by the luteinizing hormone (LH) acting through the LH/choriogonadotropin receptor (LHCGR) to stimulate androgen secretion in the interstitial Leydig cells. However, a more direct, nonsteroidal role of LH mediating the spermiogenic pathway remains unclear. Using a flatfish with semicystic spermatogenesis, in which spermatids are released into the seminiferous lobule lumen (SLL), where they develop into spermatozoa without direct contact with the supporting Sertoli cells, we show that haploid spermatids express the homolog of the tetrapod LHCGR (Lhcgrba). Both native Lh and intramuscularly injected His-tagged recombinant Lh (rLh) are immunodetected bound to the Lhcgrba of free spermatids in the SLL, showing that circulating gonadotropin can reach the intratubular compartment. In vitro incubation of flatfish spermatids isolated from the SLL with rLh specifically promotes their differentiation into spermatozoa, whereas recombinant follicle-stimulating hormone and steroid hormones are ineffective. Using a repertoire of molecular markers and inhibitors, we find that the Lh-Lhcgrba induction of spermiogenesis is mediated through a cAMP/PKA signaling pathway that initiates the transcription of genes potentially involved in the function of spermatozoa. We further show that Lhcgrba expression in germ cells also occurs in distantly related fishes, suggesting this feature is likely conserved in teleosts regardless of the type of germ cell development. These data reveal a role of LH in vertebrate germ cells, whereby a Lhcgrba-activated signaling cascade in haploid spermatids directs gene expression and the progression of spermiogenesis., Competing Interests: The authors declare no conflict of interest.

The Senegalese sole (Solea senegalensis) is a marine flatfish of high economic value and a target species for aquaculture. The efforts to reproduce this species in captivity have been hampered by the fact that farmed males (F1) often show lower sperm production and fertilization capacity than wild-type males (F0). Our knowledge on spermatogenesis is however limited to a few studies. In a previous work, we identified by 2-D DIGE several potential protein markers in testis for the poor reproductive performance of F1 males. Therefore, the objectives of the present study were, first, to investigate changes in genes and proteins expressed in the testis throughout spermatogenesis in F0 males by using a combination of transcriptomic and proteomic approaches and, second, to further compare the testis proteome between late spermatogenic stages of F0 and F1 fish to identify potential indicators of hampered reproductive performance in F1 fish. We identified approximately 400 genes and 49 proteins that are differentially expressed during the progression of spermatogenesis and that participate in processes such as transcriptional activation, the ubiquitin-proteasome system, sperm maturation and motility or cytoskeletal remodeling. Interestingly, a number of these proteins differed in abundance between F0 and F1 fish, pointing toward alterations in cytoskeleton, sperm motility, the ubiquitin-proteasome system and the redox state during spermiogenesis as possible causes for the decreased fertility of F1 fish., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pituitary gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of vertebrate reproduction. However, in teleosts with testis of semi-cystic type and asynchronous spermatogenesis, as the flatfish Senegalese sole (Solea senegalensis), the physiological roles of FSH and LH are still not well understood. To gain insight into this mechanism, full-length complementary DNAs (cDNAs) encoding Senegalese sole FSH beta and LH beta subunits, and the common glycoprotein alpha subunit (CG alpha), were cloned and sequenced. The three cDNAs consisted of 550, 582 and 744 nucleotides encoding peptides of 120, 148 and 132 amino acids, respectively. Comparison of the deduced amino acid sequences of sole FSH beta, LH beta and CG alpha with those from other teleosts indicated that cysteine residues and potential N-linked glycosylation sites were fully conserved with respect to other percomorphs and salmonids. However, the primary structure of FSH beta and LH beta in pleuronectiforms appeared to be highly divergent. In situ hybridization of mature male pituitaries showed that fshb, lhb and cga mRNAs were localized in the proximal pars distalis and in the periphery of pars intermedia. Real-time quantitative reverse transcription-polymerase chain reaction indicated that the levels of all three transcripts in the pituitary of males increased during winter and spring, at the time when plasma levels of androgens raised and testicular germ cell development and spermatozoa production were stimulated. These results suggest that FSH and LH may regulate spermatogenesis in Senegalese sole similarly to that described for other teleosts with testis of cystic type and synchronous germ cell development.

The effect of 11-ketoandrostenedione (OA) on plasma concentrations of sexual steroids and spermatogenesis of Senegalese sole (Solea senegalensis) implanted with gonadotropin-releasing hormone agonist (GnRHa) was investigated. Males were treated with saline (control) or with GnRHa implants (50 mug kg(-1)) in the presence or absence of OA (2 or 7 mg kg(-1)) during twenty eight days. Treatment with GnRHa alone slightly stimulated spermatogenesis and milt production with respect to controls, and this was associated with a transient elevation of plasma 11-ketotestosterone (11-KT) at day seven and an increase of 5beta-reduced metabolite(s) of 17,20beta-dihydroxy-pregn-4-en-3-one (17,20betaP) at day twenty eight. However, treatment with GnRHa+OA increased plasma concentrations of 11-KT and free+sulphated 5beta-reduced metabolites of 17,20betaP at days seven, fourteen and twenty one. After twenty eight days, the testis of GnRHa+OA-treated fish showed a lower number of spermatogonia B and spermatocytes I, and a higher number of spermatids, than fish treated with GnRHa alone. In addition, the motility of spermatozoa produced by GnRHa+OA males was enhanced by 2-fold with respect to controls or GnRHa males. These results suggest that treatment of Senegalese sole with GnRHa+OA stimulates spermatogenesis resulting in more motile sperm. Such effects could be mediated by an increased synthesis of 11-KT and/or 17,20betaP in the testis but further studies will be required to elucidate the specific mechanism involved.

Abstract: Aquaporin‐mediated fluid transport in the mammalian efferent duct and epididymis is believed to play a role in sperm maturation and concentration. In fish, such as the marine teleost gilthead seabream (Sparus aurata), the control of fluid homeostasis in the spermatic duct seems also to be crucial for male fertility, but no information exists on the expression and distribution of aquaporins. In this study, reverse transcriptase‐polymerase chain reaction and immunoblotting analyses, employing available and newly raised paralog‐specific antibodies for seabream aquaporins, indicate that up to nine functional aquaporins, Aqp0a, ‐1aa, ‐1ab, ‐3a, ‐4a, ‐7, ‐8bb, ‐9b and ‐10b, are expressed in the spermatic duct. Immunolocalization of the channels in the resting spermatic duct reveals that Aqp0a, ‐1aa, ‐4a, ‐7 and ‐10b are expressed in the monolayered luminal epithelium, Aqp8b and ‐9b in smooth muscle fibers, and Aqp1ab and ‐3a in different interstitial lamina cells. In the epithelial cells, Aqp0a and ‐1aa are localized in the short apical microvilli, and Aqp4a and ‐10b show apical and basolateral staining, whereas Aqp7 is solely detected in vesicular compartments. Upon spermiation, an elongation of the epithelial cells sterocilia, as well as the folding of the epithelium, is observed. At this stage, single‐ and double‐immunostaining, using two aquaporin paralogs or the Na+/K+‐ATPase membrane marker, indicate that Aqp1ab, ‐3a, ‐7, ‐8bb and ‐9b staining remains unchanged, whereas in epithelial cells Aqp1aa translation is supressed, Aqp4a internalizes, and Aqp0a and ‐10b accumulate in the apical, lateral and basal plasma membrane. These findings uncover a cell type‐ and region‐specific distribution of multiple aquaporins in the piscine spermatic duct, which shares conserved features of the mammalian system. The data therefore suggest that aquaporins may play different roles in the regulation of fluid homeostasis and sperm maturation in the male reproductive tract of fish. [ABSTRACT FROM AUTHOR]

Captive flatfishes, such as the Senegalese sole, typically produce very low volumes of sperm. This situation is particularly prevalent in the first generation (F1) of reared sole males, which limits the development of artificial fertilization methods and the implementation of selective breeding programs. In this study, we investigated whether combined treatments with homologous recombinant follicle-stimulating (rFsh) and luteinizing (rLh) hormones, produced in a mammalian host system, could stimulate spermatogenesis and enhance sperm production in Senegalese sole F1 males. In an initial autumn/winter experiment, weekly intramuscular injections with increasing doses of rFsh over 9 weeks resulted in the stimulation of gonad weight, androgen release, germ cell proliferation and entry into meiosis, and the expression of different spermatogenesis-related genes, whereas a subsequent single rLh injection potentiated spermatozoa differentiation. In a second late winter/spring trial corresponding to the sole’s natural prespawning and spawning periods, we tested the effect of repeated rLh injections on the amount and quality of sperm produced by males previously treated with rFsh for 4, 6, 8 or 10 weeks. These latter results showed that the combination of rFsh and rLh treatments could increase sperm production up to 7 times, and slightly improve the motility of the spermatozoa, although a high variability in the response was found. However, sustained administration of rFsh during spawning markedly diminished Leydig cell survival and the steroidogenic potential of the testis. These data suggest that in vivo application of rFsh and rLh is effective at stimulating spermatogenesis and sperm production in Senegalese sole F1 males, setting the basis for the future establishment of recombinant gonadotropin-based hormone therapies to ameliorate reproductive dysfunctions of this species. [ABSTRACT FROM AUTHOR]

Consecutive treatments with recombinant follicle-stimulating and luteinizing hormones (rFsh and rLh, respectively) stimulate spermatogenesis and potentiate sperm production in pubescent specimens of the oligospermic Senegalese sole ( Solea senegalensis ). However, sperm production in response to the hormones is highly variable, and the steroidogenic potential of the testis may be diminished due to sustained hormone supply. Here, we compared the effectiveness of low (9 μg/kg) and high (18 μg/kg) doses of rFsh and rLh to improve sperm production in adult sole during late winter-early spring (onset of the natural spawning period), and in autumn under a controlled temperature of 12 °C (period of testicular recrudescence). Treatment with rFsh over six weeks during spring, followed by a single rLh injection, did not enhance sperm production, possibly because of an advanced stage of sexual maturation of the males, as reflected by high Lh plasma levels (~17 ng/ml) before rFsh treatment. In contrast, in autumn, when the Lh circulating levels were much lower (~3 ng/ml), the low doses of rFsh and rLh generated a four-times increase in sperm production, whereas the high doses of the hormones were ineffective. However, treatment with rLh, regardless of the effect of rFsh, improved the motility of spermatozoa during both spring and autumn. These data confirm that consecutive rFsh and rLh treatments increase sperm production and quality in adult sole males, although they seem to be highly sensitive to the rFsh dose. The efficiency of recombinant gonadotropins also appears to be season-dependent despite the asynchronous nature of the sole testis. [ABSTRACT FROM AUTHOR]

The intensive culture of the Senegalese sole ( Solea senegalensis ) is hampered by the low or null fertilization rates exhibited by the first generation (F1) of reared males. To investigate the regulation of the reproductive processes in this species by the pituitary gonadotropins follicle-stimulating and luteinizing hormones (Fsh and Lh, respectively), we developed a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for Lh measurements. Quantification of the Fsh and Lh plasma levels in cultured sole using the Lh ELISA developed here, and a previously developed ELISA for Fsh, indicated that in both males and females circulating Fsh steadily increased during autumn and winter and prior to the major spawning in spring, whereas an Lh surge occurred specifically during spawning. The increase in Fsh was associated with a rise of plasma levels of the steroid hormones testosterone (T), 11-ketotestosterone (11-KT) and estradiol-17β (E2), but that of Lh was concomitant with a strong decline of the levels of E2 in females and of 11-KT in males, possibly reflecting a rapid steroidogenic shift promoting the final maturation of gametes. Comparison of the plasma levels of gonadotropins and steroids between wild and F1 fish during autumn and spring revealed that F1 males showed significantly lower plasma Lh titres compared to wild males, whereas the levels of T and 11-KT were similar or more elevated in the F1 fish. These data suggest that an impaired Lh secretion during spawning, and perhaps altered Lh-mediated mechanisms in the testis, may be underlying causes for the low reproductive performance of Senegalese sole F1 males. [ABSTRACT FROM AUTHOR]

In flatfishes with asynchronous and semicystic spermatogenesis, such as the Senegalese sole ( Solea senegalensis ), the specific roles of the pituitary gonadotropins during germ cell development, particularly of the follicle-stimulating hormone (Fsh), are still largely unknown in part due to the lack of homologous immunoassays for this hormone. In this study, an enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh was developed by generating a rabbit antiserum against a recombinant chimeric single-chain Fsh molecule (rFsh-C) produced by the yeast Pichia pastoris . The rFsh-C N- and C-termini were formed by the mature sole Fsh β subunit (Fshβ) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshβ-specific antibodies, which were used to develop the ELISA using the rFsh-C for the standard curve. The sensitivity of the assay was 10 and 50 pg/ml for Fsh measurement in plasma and pituitary, respectively, and the cross-reactivity with a homologous recombinant single-chain luteinizing hormone was 1%. The standard curve for rFsh-C paralleled those of serially diluted plasma and pituitary extracts of other flatfishes, such as the Atlantic halibut, common sole and turbot. In Senegalese sole males, the highest plasma Fsh levels were found during early spermatogenesis but declined during enhanced spermiation, as found in teleosts with cystic spermatogenesis. In pubertal males, however, the circulating Fsh levels were as high as in adult spermiating fish, but interestingly the Fsh receptor in the developing testis containing only spermatogonia was expressed in Leydig cells but not in the primordial Sertoli cells. These results indicate that a recombinant chimeric Fsh can be used to generate specific antibodies against the Fshβ subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes. [ABSTRACT FROM AUTHOR]

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. • Treatment with rFsh and rLh at 12 °C enhance spermiation in Senegalese sole F1 males. • One batch of spermatids is recruited into spermatozoa differentiation after a single rLh injection. • Maximum sperm production occurs 48 h after rLh injection at 12 °C. • rFsh and rLh treatments at 12 °C and 17 °C, respectively, increase spermiation. [ABSTRACT FROM AUTHOR]

Abstract: This study was aimed to investigate whether sexual maturation of immature male eels could be stimulated indirectly by placing them in contact with either male (Minj) or female (Finj) eels in which sexual maturation had been stimulated directly by weekly injections of human chorionic gonadotropin (hCG) or salmon pituitary extract (SPE), respectively. Untreated males were placed either in the same tank or in a separate tank that was linked to the injected fish via a recirculation system. The hormonal treatments stimulated spermatogenesis and spermiation in Minj, and ovulation in Finj as well as an increase of the ocular (I o) and gonadosomatic (GSI) indices in both sexes. Plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) increased in Minj and T and 17β-estradiol (E2) in Finj. A small peak of plasma 17,20β-dihydroxypregn-4-en-3-one (17,20βP) occurred during ovulation, while the plasma levels of 17α-hydroxypregn-4-ene-3,20-dione (17P) were undetectable in both males and females. The water conditioned by Minj and Finj induced significant, though relatively minor, increases in I o and GSI in uninjected males. In addition, uninjected fish showed small changes in plasma T and 11-KT levels, apparently related to the timing of spermiation and ovulation of Minj and Finj, respectively, as well as an activation of spermatogenesis (but not spermiation). Injected fish released free and conjugated T, 11-KT and E2 into the water, although immature eels were unable to smell (by electro-olfactogram) any of these steroids or prostaglandin F2α. However, immature males were highly sensitive to water extracts conditioned by spermiating Minj and pre-ovulatory and ovulated Finj. These preliminary results suggest the existence of chemical communication between maturing eels and immature males that stimulates gonad development, although the putative pheromone(s) involved has/have not yet been identified. [Copyright &y& Elsevier]