Changes of Phosphatidylcholine and Fatty Acids in Germ Cells during Testicular Maturation in Three Developmental Male Morphotypes of Macrobrachium rosenbergii Revealed by Imaging Mass Spectrometry

Testis maturation, germ cell development and function of sperm, are related to lipid composition. Phosphatidylcholines (PCs) play a key role in the structure and function of testes. As well, increases of polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA), especially arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are essential for male fertility. This study is the first report to show the composition and distribution of PCs and total fatty acids (FAs) in three groups of seminiferous tubules (STs) classified by cellular associations [i.e., A (STs with mostly early germ cells), B (STs with mostly spermatids), and C (STs with spermatozoa)], in three morphotypes of Macrobrachium rosenbergii, [i.e., small male (SM), orange claw male (OC), and blue claw male (BC)]. Thin layer chromatography exhibited levels of PCs reaching maxima in STs of group B. Imaging mass spectrometry showed remarkably high signals corresponding to PC (16:0/18:1), PC (18:0/18:2), PC (18:2/20:5), and PC (16:0/22:6) in STs of groups A and B. Moreover, most signals were detected in the early developing cells and the intertubular area, but not at the area containing spermatozoa. Finally, gas chromatography-mass spectrometry indicated that the major FAs present in the testes were composed of 14:0, 16:0, 17:0, 18:0, 16:1, 18:1, 18:2, 20:1, 20:2, 20:4, 20:5, and 22:6. The testes of OC contained the greatest amounts of these FAs while the testes of BC contained the least amounts of these FAs, and there was more EPA (20:5) in the testes of SM and OC than those in the BC. The increasing amounts of FAs in the SM and OC indicate that they are important for spermatogenesis and spermiogenesis. This knowledge will be useful in formulating diets containing PUFA and HUFA for prawn broodstocks in order to improve testis development, and lead to increased male fecundity.


Introduction
Macrobrachium rosenbergii, the giant freshwater prawn, is one of the most economically important species for global freshwater prawn farming [1][2]. Knowledge of this species, especially in nutrition and reproduction, has been acquired but remains incomplete. Consequently, prawn farmers usually face many problems during culture of the animals, imbalanced lipid consumption is a common cause of low fecundity of males in many broodstock species [3][4][5][6].
In crustacean females, there are a number of reports on lipid profiles in the ovaries, and these have been used as key knowledge to formulate balanced lipid diets. For example, ovarian lipid compositions, especially triacylglycerols (TAGs) and phospholipids (PLs), have been determined for Serolis pagenstecheri [7], Serolis cornuta [7], Penaeus monodon [8], Penaeus semisulcatus [9], M. rosenbergii [10], Litopenaeus vannamei [11], Fenneropenaeus indicus [12], Cherax quadricarinatus [13], Portunus sanguinolentus [14], Albunea symmysta [15], and Penaeus merguiensis [16], and indicated that lipid changes are associated with ovarian maturation and embryonic development. This has provided data for formulated balanced lipid diets for females. On the other hand, studies in males have focused on testicular lipids, including TAGs and PLs, in S. pagenstecheri [7], S. cornuta [7], Pleoticus muelleri [17], P. monodon [8], and Macrobrachium nipponense [18]. These reports indicated that the amount of lipids in the testes were lower than the ovaries and usually contained eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). However, arachidonic acid (ARA) was found to be higher than EPA and DHA in the spermatophores of P. monodon [3]. A knowledge of lipid composition in the testes of developing males of M. rosenbergii is now needed in order to formulate balanced diets for the improvement of male fecundity.
The PLs, especially phosphatidylcholines (PCs), are major integral components of plasma membranes, and are also involved in sperm membrane permeability and fluidity [19][20][21][22], acrosomal reactions [23], and sperm motility [24]. PCs are composed of a choline head group, glycerol, and two fatty acid side chains that can be saturated and/or unsaturated. PC treatments have prevented lipid peroxidation or degradation of enzymes in stored semen of the turkey [25], and improved acrosomal responses in human sperm [23].
It has been reported that fatty acid (FA) side chains of lipid molecules, especially in polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA) play important roles in reproduction [4], [21], [26][27][28][29][30]. The three best known HUFA molecules concerned with reproduction are ARA, EPA, and DHA. ARA is a precursor of series II prostaglandins (PGs), whereas EPA is a precursor of series III PGs [31]. Both PGs are involved in steroid production [32]. The role of these two molecules and DHA has been studied in the goldfish [32], and it was found that they all control steroidogenesis in the testis, and that EPA deficiency delayed spermiation and decreased fertilization rates. For penaeid shrimps, including P. monodon and L. vannamei, it was found that the diet containing polychaetes, mollusk, squids, fish, vegetable oils which are rich in HUFA and PUFA, especially ARA, EPA, and DHA, could improve the quality of spermatophores and sperm [3,5,[33][34]. Similarly, diet containing these natural components could also enhance male reproductive performance in M. malcolmsonii [35]. Another study reported that the EPA-containing diet enhanced sperm production in the freshwater crayfish, Astacus leptodactylus [4], and HUFA was found to increase the recovery of spermatogenesis in n-3 desaturase-null mice that cannot synthesize HUFA [30].
Mammalian spermatogenesis occurs in the seminiferous tubules (STs) following puberty, which starts from mitotic divisions of type B spermatogonia into primary spermatocytes [36]. The primary spermatocytes then go through meiosis I to produce secondary spermatocytes, meiosis II to produce haploid spermatids, and transformation of spermatids into spermatozoa that contain less cytoplasm [36]. Furthermore, germ cells in STs are supported by Sertoli cells or nurse cells [36][37]. So, each mammalian ST contains a mixture of developing germ cells and spermatozoa designated as cellular association, which can be classified into 14 stages in human [38].
In contrast, the STs of M. rosenbergii have been characterized into 9 maturation stages [i.e., stages I to IX], according to cellular association [39]. Stages I to V contained mostly primary and secondary spermatocytes; Stages VI to VIII contained mostly spermatids (early, middle, and late spermatids); and Stage IX contained mostly spermatozoa with decondensed chromatin. In all stages, the nurse cells and spermatogonia were always located on the basement membrane [39]. Moreover, M. rosenbergii males have been characterized into three distinct developmental morphotypes [i.e., small male (SM), orange claw male (OC), and blue claw male (BC) with fully mature testis] [40][41]. The lipids and FAs required for maturation of the STs within the three developmental male morphotypes of M. rosenbergii have not been studied. Since each ST is too small to be analysed for lipid profiles by imaging mass spectrometry (IMS), the STs were, therefore, sub-grouped into three broad maturation groups based on cellular components [i.e., A (Stages I-V), B (Stage VI-VIII), and C (only Stage IX)].
There are several ways to reveal lipid and FA compositions, namely thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). However, these methods are not able to localize the lipid molecules in tissue sections. IMS is a new technique used to determine the distribution of all lipids contained within tissues at high resolution. Recently, our collaborative research using IMS has successfully visualized seminolipid and metabolites in mouse testes [42][43], and PCs and TAGs in ovaries of P. merguiensis [16]. However, there have been no IMS analyses of male M. rosenbergii testes.
In this study, we focused on the localization and quantification of PCs, and the composition of FAs, including PUFA and HUFA, in the testes of three developmental male morphotypes of M. rosenbergii, and in during three phases of ST maturation which contain different stages of developing germ cells. The results are now being used to produce balanced formula diets for male broodstocks, especially with appropriate contents of PUFAs and HUFAs in order to increase male fecundity.

Animals and histology of the seminiferous tubules
Thirty male giant freshwater prawns in each developmental morphotype, namely SM, OC, and BC, were obtained from a commercial farm in Suphanburi province, Thailand. The prawns were anesthetized on ice for 2 min. The testes were dissected out, (i) frozen immediately in liquid nitrogen and stored at -80°C, and (ii) fixed in 4% paraformaldehyde in 0.1 M phosphatebuffered saline (PBS; 0.033 M NaH 2 PO 4 Á2H 2 O, 0.067 M Na 2 HPO 4 ÁH 2 O, and 0.145 M NaCl), pH 7.4, to confirm the structure by paraffin section (5 μm). The frozen testes of the three male morphotypes were divided into two equal parts for cryosection and lipid extraction S1 Fig. One part of each frozen tissue (at the base only) was attached to a specimen plate using OCT compound (Optimum Cutting Temperature, Sakura Finetek 4583, Sakura, Tokyo, Japan) and sectioned (~10 μm) with a cryostat, CM 1950, (Leica Microsystems, Wetzler, Germany), after which sections were transferred to silane-coated slides (Sigma-Aldrich, Missouri, USA) for characterizing the stages of seminiferous tubules by hematoxylin and eosin (H&E) staining Fig. 1 Left column. The sections were dried using a hair dryer and stained with Mayer's hematoxylin solution for 10 min, washed with tap water, counterstained with eosin, and mounted by Permount (Bio-Optica, Milan, Italy). They were then examined under a Nikon E600 light microscope (Nikon, Tokyo, Japan), and images were captured by a Nikon DXM digital camera using an ACT-1 program.

Lipid extraction
The frozen testes of each group and stage were weighted, pulverized, and extracted with 0.1 g/ml of extraction solution (chloroform: methanol, 2:1 v/v) following the method described earlier [16,42]. The samples were then sonicated for 10 s and stopped for 5 s, and this procedure was repeated 10-15 times using a Microson, Ultrasonic Cell Disruptor XL-2000 (Wakenyaku Co. Ltd., Kyoto, Japan). The glass tubes containing the sonicated tissues were tightly wrapped with parafilm, and then incubated overnight at room temperature. The samples were centrifuged at 3000 xg for 5 min to separate the tissue residues, and the solutions containing lipid were collected and transferred to new glass tubes, wrapped, and stored at -80°C until being analysed.

Separation and quantification of lipids by thin layer chromatography (TLC)
The extracted lipids were separated with TLC using the method described earlier by our group [16,42]. The solution containing extracted lipids (3 μl per sample) and the PC standard (Sigma-Aldrich, Missouri, USA) were spotted (with each spot being 5 x 1 mm in size) onto high performance thin layer chromatography glass plates (HPTLC silica gel 60 with the size 100 x 100 mm-Merck, Darmstadt, Germany), and dried at room temperature. Each HPTLC plate was immersed in a TLC chamber containing separation buffer (methylacetate, n-propanol, chloroform, and 0.25% KCl in the ratio 25:25:10:9 v/v/v/v). Each HPTLC plate was airdried after separation, and then was sprayed with primuline reagent (Nacalai Tesque, Inc., Kyoto, Japan) composed of 1 mg of primuline in 100 ml of 80% acetone in water. After drying, the PC bands were visualized and photographed under UV light (FAS-III, Toyobo Co. Ltds, Osaka, Japan). The intensities of the bands were analysed by ImageJ software (http://rsbweb. nih.gov/ij/).

Identification of lipids by tandem mass spectrometry (MS/MS)
The extracted lipids from testicular tissues of each maturation group of STs and male morphotypes were thoroughly mixed 1:1 v/v with matrix solution (20 mg/ml DHB in 70% methanol and 0.1% TFA). Aliquots of 1 μl of the solutions were applied manually to a stainless plate and cool air-dried using a hair dryer. A calibration process was performed using 10 pmol/μl bradykinin and 10 pmol/μl human angiotensin-II as standard peptides. The MS/MS analyses were performed using a QSTAR Elite high-performance, hybrid quadrupole TOF mass spectrometer (Applied Biosystems/MSD Sciex, Foster City, CA). The extracted lipids were ionized in positive ion mode and fragmented with collision energy between 30-40 V. After being analysed, the precursor ions were identified based on neutral losses in the product ion spectra and confirmed by using Metabolite MS Search (http://www.hmdb.ca/spectra/ms/search).

Distributions of phosphatidylcholine by imaging mass spectrometry (IMS)
A part of each frozen testis (used for histology) was sectioned at 10 μm of thickness with a cryostat (CM 1950, Leica Microsystems). The sections were thaw-mounted onto indium tin oxide (ITO)-coated slides (Bruker Deltonics, Bremen, Germany), dried and then kept at -30°C until IMS analysis. Before IMS analyses, the sections were dried at room temperature and then sprayed with matrix solution using a 0.2-mm nozzle caliber airbrush (Procon Boy FWA Platinum, Tokyo, Japan). The matrix used was 2,5-dihydroxybenzoic acid (DHB) (Bruker Daltonics), and it was firstly dissolved to reach a concentration of 50 mg/ml in 70% methanol and 0.1% trifluoroacetic acid (TFA). A calibration process was performed using 10 pmol/μl bradykinin and 10 pmol/μl human angiotensin-II as standard peptides by applying on to the sprayed area out of the tissue sections. The sprayed sections were then analysed in a positive ion mode using an ultraflex II MALDI TOF/TOF mas nics). The mass spectra were obtained in the mass ranges between m/z 500-1000. The settings of laser s spectrometer (Bruker Delto irradiation were 200 Hz frequency and a raster width at 20 μm. After IMS analyses, ion images were obtained using flexImaging 2.1 software (Bruker Daltonics). Finally, the analysed sections were stained with H&E to confirm the histology of the area of interest.

Analyses of fatty acids by gas chromatography-mass spectrometry (GC-MS)
These analyses followed the methods described earlier [16]. The extracted lipids were spiked with an internal control (0.4 mg/ml arachidic acid (20:0) diluted in chloroform:methanol at a ratio of 2:1), and then dried by nitrogen gas using a TurboVap LV Evaporation System (Caliper Life Sciences, Hopkinton, MA, USA). After being completely dried, the lipids were methylated using a fatty acid methylation kit (Nacalai Tesque, Inc., Kyoto, Japan), and then purified using a fatty acid methyl ester purification kit (Nacalai Tesque, Inc.). The purified FAs were stored at -3°C until analysed by GC-MS.
The purified FAs from testes of each morphotype were separately injected (1 μl per sample) into a GC-MS QP-2010 Plus (Shimadzu Co., Kyoto, Japan), applied with a DB-5MS column (3060.25 mm I.D., 0.25 mm; D.F., Agilent technologies, CA, USA). The purified FAs were analysed under a column temperature of 210°C and column pressure between 110 kPa-380 kPa at 7 kPa/min. After analyses, the FAs were identified and the amount calculated using the internal controls as a reference.

Statistical analyses
The intensity of each band from TLC analyses and FAs amount of each testis stage and male morphotype from GC-MS analyses were expressed as a mean ± S.D. and the data was then compared using a Student's t-test to determine differences. A probability value of less than 0.05 (P<0.05) indicated a significant difference.

Histology of the seminiferous tubules
Spermatogenesis within the STs has been classified into 9 stages corresponding to the presence of different types of spermatocytes, spermatids, and spermatozoa [39]. In our research using 10 μm-thick cryosection, it was difficult to clearly identify all 9 stages of the STs. However, based on the histological outlines and abundance of spermatogonia (Sg), spermatocytes (Sc), spermatids (St), and spermatozoa (Sz) present in the tubules we could identify the stages of the STs and separated them into three groups representing early, middle, and late stages of spermatogenesis Fig. 1a-e, f-j, k-o. Group A (including stages I-V), contained Sg and nurse cells (Nc) that were located on the basement membrane, and mostly Sc Fig. 1a-e. Group B (including stages VI-VIII), contained some Sg and Sc, but mostly St and immature Sz with condensed chromatin Fig. 1f-j. Group C (stage IX), contained mostly mature Sz with de-condensed chromatin and NC, which were located close to the basement membrane Fig. 1k-o. In all stages, the STs were surrounded by intertubular area (IT) made up mainly by connective tissues. All three groups of ST stages were found in the three male morphotypes, but in different proportions. For example, SM contained mostly group A, OC contained mostly group B, and BC contained mostly group C.

Quantification of lipids by thin layer chromatography (TLC)
The extracted lipids were separated by TLC, and the highest intensity signals were found in PC bands of each group. The PCs bands were expressed as mean ± S.D. which showed different amounts in each of the ST groups Fig. 2A and the male developmental morphotypes Fig. 2B. The STs of group B which contained mostly spermatids and some immature sperms showed significantly higher intensities compared with group A and C (P<0.05) Fig. 2A. The highest amounts of PCs could be observed in the OC males, which is the transitional stage from SM to BC male, and the lowest PC amounts were observed in BC males (with significant difference at P<0.05) Fig. 2B   All signals from ion images Table 1 were identified in the same way as the two signals de-    Table 1, PC (18:1/18:2) represented by m/z 806.5 S3e Fig. and Table 1, and PC (18:0/18:1) represented by m/z 810.5 S3f Fig. and Table 1, also showed high signal intensities in the IT. (iv) Lastly, the PCs presented only in the STs of group A, comprised of PC (16:0/22:6 (DHA)) represented by m/z 828.5 and 844.5 S3j Fig. and Table 2, PC (18:0/20:4 (ARA)) represented by m/z 832.5 S3i Fig. and Table 2 Table 2, showed high signal intensities in developing germ cell areas.

Discussion
This study, using IMS and related techniques, is the first to show the localization and composition of PCs and FAs, especially HUFA and PUFA, in three maturing ST groups of the three male morphotypes of M. rosenbergii. We focused on the changes of PCs and FAs during ST maturation, and developmental stages of male morphotypes during maturation from young SM to mature BC [39]. We found that (1) STs of group B had higher amounts of PCs and FAs than groups A and C; (2) OC males always contain higher amounts of PCs and FAs (except EPA) than the SM and BC; (3) SM males contain high ratios of HUFAs and PUFAs; (4) EPA is always higher than DHA in all ST groups, and in all male morphotypes; and (5) the PCs identified in the testes of this species were considerably higher in developing germ cells and the IT.
The composition of total lipids in each developmental morphotype of M. rosenbergii has been found to be different [44]. In particular, it was found that the total lipids in the hepatopancreas, a major energy storage organ in crustaceans, to be highest in OC males and lowest in mature BC males. This result supports our TLC results that showed trends of PC amounts in the three developmental morphotypes Fig. 2B. The OC males are reproductively less active than BC males, but are growing more rapidly than young SM and mature BC males [44][45][46][47]. Surprisingly, in OC males the group B STs with differentiating Sts had higher levels of PCs than group C STs that contain only spermatozoa Fig. 2A. It was reported that the decrease of lipid levels in the testes of BC males may relate to germ cell developmental processes in which there is an extrusion of numerous cytoplasmic components including lipids as they become mature spermatozoa [44][45][46][47]. This also supports our results as the lowest amounts of PCs and FAs were detected in the testes of BC males Figs. 2 and 6A.
IMS is a powerful technique to reveal the location of the lipids such as PCs, phosphatidylinositols, phosphatidylethanolamines, seminolipids, and TAGs in the reproductive organs of mice and shrimps, without contamination that may be introduced with embedding media [16,42]. Recently, Goto-Inoue (2012) reported that lipids changed during testis maturation in mice, especially lipids in the positive ion mode detected in the range of m/z 700-900, with substantial signals corresponding to PCs. The highest intensity of these was found at m/z 798.5 [PC (16:0/18:1) + K] + [43]. Moreover, Chansela (2012) reported that there were relatively large amounts of PCs in the ovary of P. merguensis [16]. Our IMS results of the testis of M. rosenbergii showed numerous signals corresponding to PCs, which included HUFA-containing PCs with m/z 820. type of developing male germ cells were found to be different. In mammals, PUFA and the major HUFA (namely DHA) accumulated at highest levels in cell membranes of male germ cells, and were essential for male fertility [48][49]. In rats, the spermatids contain more docosapentaenoic acid (DPA)-containing phospholipids than spermatocytes [50], indicating that there are species differences in the types of lipids, and qualities of developing male germ cells, implying their importance during differentiation.
Furthermore, the levels of HUFA in the male of this species are decreasing in the testes of the blue claw males which contained mostly mature sperm cells with small membranes of early germ cells (Sg, Sc). In SM and OC, the testes contain large amounts of developing germ cells in the spermatogenic zone (in STs of groups A, B), which is highly active in spermtogenesis [52]. After maturation, the testes of BC males contain much thinner spermatogenic zone, and mature Sz, thus the STs function is more in the storage of Sz rather than producing Sz [52]. In addition the Sz of this prawn are immobile due to the lack of tail and the nuclear chromatin is totally decondensed [39]. They are thus relatively inert compared to the mammalian sperm. It is possible that their membranes are less fluid and need much less HUFA when they reach complete maturity.
Finally, we recommend that diets containing lipids with high levels of HUFA, PUFA, especially EPA and DHA, should be given to the SM males for improving germ cell development and increase energy accumulation to shorten their developmental processes. This knowledge could be useful in formulating suitable nutrition to each male morphotype broodstock of M. rosenbergii, which is important commercial species in freshwater prawn farming countries.