Reproduction in Aquatic Animals

### **Chapter 7**

## Stimulatory Effects of Androgens on Eel Primary Ovarian Development - from Phenotypes to Genotypes

*Yung-Sen Huang and Chung-Yen Lin*

### **Abstract**

Androgens stimulate primary ovarian development in Vertebrate. Japanese eels underwent operation to sample the pre- and post-treated ovarian tissues from the same individual. Ovarian phenotypic or genotypic data were mined in a pair. A correlation between the initial ovarian status (determined by kernel density estimation (KDE), presented as a probability density of oocyte size) and the consequence of androgen (17MT) treatment (change in ovary) has been showed. The initial ovarian status appeared to be important to influence ovarian androgenic sensitivity. The initial ovary was important to the outcomes of androgen treatments, and ePAV (expression presence-absence variation) is existing in Japanese eel by analyze DEGs; core, unique, or accessory genes were identified, the sensitivities of initial ovaries were correlated with their gene expression profiles. We speculated the importance of genetic differential expression on the variations of phenotypes by 17MT, and transcriptomic approach seems to allow extracting multiple layers of genomic data.

**Keywords:** androgen, *Anguilla japonica*, ePAV, kernel density estimation, primary ovary, transcriptome

### **1. Introduction**

Anguilla eels, *Anguilla spp.*, are important economic species, but eel seeds (glass eels) for aquaculture are obtained only from the wild. Moreover, the resource of glass eels has been drastically reduced since the 1970s. The artificial propagation of eel seed is demanded. The complex life cycle of Anguilla eels, undergo several metamorphoses and migrate between freshwater and ocean environments, leads an obstacle to the artificial propagation on a commercial scale, although progress in the artificial maturation of eel broodstocks and larvae rearing has been achieved for Japanese eels [1–3]. Indeed, to obtain eel seeds in captivity, an important factor is the selection of broodstocks for artificially maturation [2]. It has been demonstrated that wild silver eels (undergoing pubertal metamorphosis) are the best candidates for artificial maturation [4]. However, natural sources of wild female silver eels are diminishing.

There are different responsive abilities of different eels on piscine pituitary extracts (PPE) in artificial maturation manipulation. This phenomenon has been commented: "the maturity status of parental females at the beginning of artificial maturation trials is also an important factor affecting the success of maturation experiments" [2] as well as suggested that silver eels with more developed oocytes (mean of oocyte diameter is 0.262 mm) required a shorter time to produce mature eggs than did cultivated eels (mean of that is 0.177 mm) [4], the maturity of morphological changes (silvering, as a pubertal metamorphosis in eels) might have a close correlation with ovarian development (oocyte growth). The physiological status of the eels just before PPE injection has been postulated to be a reason for this difference [3]. In addition, in most circumstances during artificial eel maturation, unsynchronized development stages of ovary are a problem, androgens have been demonstrated to synchronize oocyte development in the eel ([5] references therein). While, in practice, the stimulatory effect of androgens (or PPE) on eel ovarian development was often not uniform or not conserved among different individuals, and the stimulatory effects might depend on the initial status of ovarian development [3, 4]. Artificial maturation in eels appears to be paralogue to 'personalized medicine' in human diseases. Identifying a set of gene biomarkers to predict or estimate the outcomes of eel artificial maturation is interesting and is demanded.

### **2. From phenotypes**

Indeed, the developmental stages of oocytes in Japanese eels were classified by the mean of oocyte diameter as follows: oil droplet stage for follicles <200 μm; early vitellogenic stage for follicles 200–400 μm; mid-vitellogenic stage for follicles 400–600 μm; late vitellogenic stage for follicles 600–800 μm; and migratory nucleus stage for follicles 800–1000 μm [4, 6].

Androgens stimulate the development of the preantral follicle in mammals [7]. In fish, androgens participate in the promotion of primary and/or early secondary ovarian follicle growth (reviewed by [8]), the primary and/or early secondary ovarian follicle stages in the fish is analogous to the preantral follicle in mammals. The stimulatory effect of androgens has been reported in coho salmon (*Oncorhynchus kisutch*) *in vivo* [8]. Androgens also significantly promote primary growth (PG) and the pre-vitellogenic (PV) transition in eels *in vivo* (*Anguilla japonica*: [9]; *Anguilla australis*: [10]). Furthermore, androgen treatment is recommended prior to or during eel artificial maturation (*Anguilla japonica*: [5] references therein; *Anguilla australis*: [10]; *Anguilla anguilla*: [11]).

Indeed, artificial maturation is often unsuccessful when the eel broodstocks come from aquafarms or have a larger body size [12]. Whether, the hypotheses that the outcomes of artificial eel maturation are based on "the maturity status of parental females at the beginning of artificial maturation trials" [4] and "the physiological status of the eels just before hormone" [3] are accepted. Therefore, identifying sex and evaluating gonadal maturity are necessary.

A method to track ovarian development before- and after-androgen (17α-methyltestosterone, 17MT) treatment in the same eel has been developed [5]. It must be emphasized that the experimental data came from the same individual; this traceability was the advantage, but the bias caused by a single sample could not be avoided. The pretreated ovary was used as a morphological or molecular baseline to compare the posttreatment ovary. Since the experimental eels and PPE were both

### *Stimulatory Effects of Androgens on Eel Primary Ovarian Development - from Phenotypes… DOI: http://dx.doi.org/10.5772/intechopen.99582*

from wild populations and subject to important individual variation, both of them are not consisted. This means the results based on two important variations if the different eels as well as PPE were employed simultaneously. To increase sample size can reduce the variation (to approach normal distribution), it can be speculated that the normal distribution is composed of many eels, the number of eels will be numerous, when one eel was selected, since time cannot be reversal, the normal distribution will collapse into one sample, then the destinations or outcomes of the selected one might be an another distribution (not always a normal distribution), but, apparently, there should be a closer correlation between the inputs and the outcomes. The data followed this protocol can be accumulated then be clustered, different patterns or clusters related with certain biological outcomes may be distinguished.

Eels were surgically operated to identify sex and to sample ovarian tissues, the operated eels recovered and the sutures were shed after 4–5 weeks [5, 13]. The manipulation did not suspend ovarian development under the subsequent artificial maturation regimen.

Primary ovarian sensitivity to androgens (17MT) is well documented; this characteristic was used to study the correlation between basal ovarian status and the outcome in Japanese eel. The lard grease- and Vaseline-based sustained-release mixtures were used *in vivo* to stimulate ovarian development. The recovered eels were treated with lard grease-Vaseline +17MT (LVMT) for four weeks. Classically, the ovarian status was estimated by the measurement of the oocyte size in the sampled ovarian tissue, then the distribution of oocyte size was plotted in histogram, histogram is a description method, the mean (m) and standard error (SE) of oocyte size were calculated (e.g. [4]). Kernel density estimation (KDE), a nonparametric way to estimate the probability density function of a random variable, was used to evaluate the ovarian status; one of the advantages of KDE is, compared with histogram method, the tiny differential varieties on the ovarian status can be percept; indeed, the measurement of oocyte diameter under microscope is a daunt task, many conditions can aberrate the results, the oocytes might be deformed by the manipulation of sampling process and tissue fixed. The oocyte diameter was derived from digital data (virtual measurements), we could not deny that the fidelity of 'real' oocyte diameter

#### **Figure 1.**

*Mean oocyte diameters of different individuals treated with LVMT. Open or solid columns represent pretreatment or posttreatment, respectively. Different letters represent significant differences (p < 0.05) among different data.*

### **Figure 2.**

*Estimation of ovarian status by kernel density estimation (KDE). The x-axis and y-axis represent the oocyte diameter (mm) and probability density, respectively. 0.1 mm was chosen as an arbitrary boundary. The distribution of oocyte sizes from the eel no 2003, no 1440 , and 2613 are presented in (a), (b), and (c), respectively.*

*Stimulatory Effects of Androgens on Eel Primary Ovarian Development - from Phenotypes… DOI: http://dx.doi.org/10.5772/intechopen.99582*

might be lost and erratic, but this shortcoming could be ameliorated by KDE, Kernel smoothing refers to a general methodology for the recovery of underlying structures in datasets [14].

In the pretreatment eels, there was no significant (p > 0.05) difference among them on mean of oocyte diameter, the developmental stage of our experimental eels was the oil droplet stage (primary oocyte). After 4 weeks of treatment (posttreatment), The mean diameter of oocyte significantly (p < 0.05) increased in the same individual, and the mean of fold change was x1.25. Based on KDE and using 0.1 mm as the arbitrary boundary, the percentage of the oocyte diameter over 0.1 mm increased from 44–70% [5].

Three eels, with different fold change on oocyte diameter x1.30, x2.08, and x2.35 tagged with no 2003, no 1440, and no 2613, respectively, were chosen to present the different results of the17MT treatment, although the growth of all oocytes was significantly (p < 0.05) stimulated by LVMT, as revealed by pre- vs. posttreatment comparisons (**Figure 1**). Moreover, analyzed by KDE, and 0.1 mm was used as an arbitrary boundary to evaluate ovarian status, the percentage of the oocytes over 0.1 mm increased from 22–60% in eel no 2003, from 42–90% in eel no 1440, and from 40–89% in eel no 2613. It seemed that oocyte growth (development) was more synchronized in eel no 2613 than in eel no 1440 because the bandwidth in eel no 2613 (bandwidth = 0.009722) was smaller than that in eel no 1440 (bandwidth = 0.02096), which indicates a more dispersed trend in ovarian development in eel no 1440; these data corresponded to the standard deviations (SDs) of 0.0574 and 0.0352 for eel no 1440 and no 2613, respectively (**Figure 2**) [5].

KDE may be a way to mine the "maturity status" of the ovary. From plotted figure, if we chose 0.1 mm as the boundary, we can speculate that if the main peak was located on or right-hand to the boundary, better oocyte development results could be detected under the subsequent treatment condition with 17MT.

More than two peaks can identified in certain plotted figures, a shoulder instead of a smooth bell curve was also observed, hidden peak was likely present [5]. Our observed data implied a wave-like on the development of Japanese eel oocytes [5]; coincidently, the Japanese eel might spawn more than once during a spawning season [15]. Moreover, there is also an argument that if the Japanese eel is a not semelparous (monocyclic) fish [16].

The pretreated ovary was used as a morphological or molecular baseline to compare the posttreatment ovary in the same individual. The comparison was based on the pattern from a probability density function (KDE); the patterns represented ovarian status. Our results showed that a different ovarian status seemed to lead to a different artificial maturation results [5].

### **3. To genotypes**

In Japanese eel, the correlations between the initial ovarian status, represented by the probability distributions of oocyte diameter, and the effects of 17MT treatment is shown [5]. We continued to investigate the relationships between the initial ovarian gene expression profiles and the stimulatory effects of androgens on oocyte development in eels. As the activation and development of ovaries are sophisticated (reviewed by [17, 18]), myriad factors and ample genes interact with each other to

control folliculogenesis [19]. Following this concept, we studied this phenomenon based on the ovarian expression profiles of the initial ovaries using transcriptomic tools, high-throughput methods for the large-scale analysis of ovarian gene functions can be used to approach this question.

Gene set analysis (GSA) has the advantage of incorporating existing biological knowledge into the expression analysis (reviewed by [20], references therein). Gene Ontology (GO) terms can be used to define gene sets, and GO analysis is a standard way to define the relationships among expressed genes, thus enabling identification through the use of GSA. Both GO and COG (Cluster of Orthologous Genes) provide specific information about genes or gene products.

In teleosts, there is a lack of transcriptome analysis on early folliculogenesis (reviewed by [21]). In coho salmon (*Oncorhynchus kisutch*), 11-ketotestosterone promoted the growth of primary ovarian follicles and altered the ovarian transcriptome dramatically [8]. In Japanese eel, ovarian transcriptomic information from eels treated with salmon pituitary extracts has been reported [22].

The number of total trinity transcripts was 541,020, and 452,778 transcripts were derived from raw assembled transcripts but clustered with 95% similarity. The number of total trinity 'genes' was 336,973, while a total of 25,340 unigenes were distributed into the 3 categories with 37.56% in biological processes, 49.15% in molecular functions, and 13.28% in cellular components. Meanwhile, the top 10 genes in biological processes, cellular component, and molecular function were indicated. According to the annotation of COG (clusters of orthologous groups of proteins), these genes were classified into 25 different functional classes. The cluster for general function prediction (19,089, 24.77%) represented the largest group, followed by cell cycle control, cell division, chromosome partitioning (11,670, 15.14%), replication, recombination and repair (6,704; 8.70%), and unknown functions (5,286; 6.86%) [23].

The post-treatment ovarian tissue was compared with the initial ovarian tissue in the same eel. The results showed that certain pathways were significantly (*p* < 0.05) upregulated, but in the same time, the same pathways were also significantly (*p* < 0.05) downregulated. These contradictory results might imply the complexity of gene network, indeed, abundant mRNA isoforms in fish seems to play important roles on this situation (the discussion further below: 4. From ePAV to phenotypes). GSA uses all of the available gene expression data (cutoff-free) instead of pre-filtering (cutoff-based) the data for a list of strong DEGs. Our cutoff-free results indicated that most of the sequenced genes were responsible for fundamental biological regulation and metabolism. Anyways, significant metabolic activities corresponded with the change in ovarian status is indicated [23].

REViGO (http://revigo.irb.hr) was used to summarize lists of GO terms by finding a representative subset of the terms. The analysis was based on the post-treatment ovarian tissue compared with the initial ovary (e.g., 2003MT/2003). The total GO enrichment score of eels 2003, 2613, and 1440 was 184, 253, and 230, respectively. These results corresponded with the phenotypes. The outcomes from the GO enrichment analysis and phenotypes were mutually supported and appeared to have a positive correlation. The ovarian gene expression profiles from the same eel before-and after-treatment are investigated. The profiles of the heatmap or cluster indicated that eel 2613 and eel 1440 were under the same clade (cluster), and eel 2003 appeared to be isolated from both. And, the gene expression profiles were more discrete in eel 2613 between the initial ovary and post-treated ovary compared with those of the two other eels (**Figure 3**). Plotting the TMM expression of all samples and calculating the Pearson correlation

*Stimulatory Effects of Androgens on Eel Primary Ovarian Development - from Phenotypes… DOI: http://dx.doi.org/10.5772/intechopen.99582*

**Figure 3.** *Gene expression levels heatmap among different individuals. Clusters of genes with different expression values were indicated.*

coefficient, the coefficient was 0.34, 0.17, and 0.15 for eel 2003, 1440, and 2613, respectively, between the initial ovary and post-treated one in the same individual [23].

The Principal Component Analysis (PCA), the distance between any given two points represents their divergences in the chart, results are also shown: the distance between eels 1440MT and 2613MT was short on PC1 (X-axis), while eel 2003MT was isolated on the other side; eel 26130MT was closer to 2003MT while 1440MT was located from the other two on PC2 (Y-axis). The order of the intra-group distance was: 2613 > 1440 > 2003; the major shift was along PC1, and there was slight shift along PC2 except for eel 2613. Moreover, the results from the heatmap also indicated that eels 2613MT and 1440MT under the same clade (cluster), but eel 2003MT was isolated from both. The results are supported mutually (**Figure 4**) [23].

We preferred to mine the differences in gene expression profiles, thoroughly as these differences might provide the bases to set up biomarkers. To maximize the differences significantly, the cutoff condition was set with the TPM of the gene as zero in the initial ovarian tissues. We attempted to filter the differences between the sensitive group (2613MT and 1440MT) and insensitive one (2003MT), the TPM was set to be equal or greater than 0.01 (TPM ≥ 0.01) to amplify the differences; there were 467 genes exclusively in 2613MT and 518 genes exclusively in 1440MT. As there were many isoforms, the number of unique genes was 266 and 303 for 2613MT and 1440MT, respectively. These unique genes are involved in various biological pathways. There were many GO enrichment biological pathways in addition to housekeeping metabolic pathways, the neurogenesis pathways (axon guidance, eye morphogenesis, and axon development in 2613MT; sensory organ development, regulation of synapse organization, and regulation of nervous system development in 1440MT) appeared to overlap in these two eels. These data implied plausible roles of neurogenesis in the primary development of the ovary [23].

### **Figure 4.**

*Analysis gene expression profiles by PCA. The initial ovaries or the post-treated ones were symbolized as open pentagon or open triangle, respectively; the arrowhead represent the change of gene profiles.*

The cutoff levels were TPM ≥ 0.01 and TPM = 0 for the sensitive group and insensitive group, respectively, tried to filter data based on DEGs to maximize the differences. Our assumption was that the significant (≥ 0.01 *vs* 0.00) DEGs among the initial ovaries might be the basis for the outcomes of 17MT subsequently, and certain subtle pathways might be amplified; this might be arbitrary and abstract, since it is impossible to interpret the information from a transcriptome without the context of experiment.

Interestingly, several pathways implicated in neurogenesis or neuro-activity have been mined. We changed the cutoff-levels; curiously, the pathways implicating eyes or sensory organ development were found. The retina is the most accessible part of the vertebrate central nervous system, and the neural retina constitutes an excellent system with which to analyze key aspects of neurogenesis [24]. Moreover, it is well recognized that the silver eel (the prepubertal stage) has larger eyes, and a similar situation might occur in the developing gonads. Our cutoff-based results led us to postulate that neurogenesis and/or neuro-activity is involved in primary ovarian development in Japanese eel. This hypothesis might pertain to the fact that in eel, even entering the silver stage, the reproductive endocrine system is blocked, but a slight development of ovary is observed.

Few reports have discussed the involvement of neurogenesis in primary ovarian development in fish. Meanwhile, in mammals, the development of ovarian nerves precedes the onset of folliculogenesis [25], and the neural activity might be an important factor in the regulation of follicular development before the ovary acquires

### *Stimulatory Effects of Androgens on Eel Primary Ovarian Development - from Phenotypes… DOI: http://dx.doi.org/10.5772/intechopen.99582*

responsiveness to gonadotropins [26], the developing ovary is regulated by direct sympathetic inputs which function in addition but complementarily to gonadotropin [27]. On the other hand, the density of innervation may contribute to the selection of follicles for further development (reviewed by [28]). Evidence supports complementary control performed by sympathetic nerves innervating the ovary [29, 30]. Moreover, our non-*de novo* data (the sequence reads were compared with a reference genome and assembled) showed that the expression levels of ovarian doublecortin (DCX), an important marker of neurogenesis for comparative neurobiologists [31]. In summary, ovarian development in all vertebrates appears to be under at least partial neural control [32], and the neural–ovarian connection established during postnatal development is necessary [33].

Indeed, our non-*de novo* data indicated that the expression levels of ovarian *FSHR* were higher in eel 2003MT, but that in 1440MT and 2613MT was below detectable levels; it is interesting that the fold-change order of *FSHR* expression between the initial ovary and the post ovary was 2613 > 1440 > 2003. These results correspond with a stimulatory effect of androgens on ovarian *FSHR* expression levels in eels [34] or in mice [35]. This data also indicated the importance of pair-sample design in genetic studies.

Biomarkers are biological characteristics that predict treatment responses. The sensitivity of oocytes to 17MT is dependent on the ovarian status (not the mean size of the oocyte) [5]. From our results, it seems not ideal to select certain genes based on DEGs to serve as biomarkers, instead of independent genes with significant DEGs, a set of genes in certain pathways to represent the ovarian status seems chosen. The initial ovarian status is fundamental to influencing how genotypes are translated into phenotypes by treatments. It is already useful to inspect the size of sampled oocytes, while our report indicated that, the gene expression pattern can ameliorate and improve the method based on phenotype for the eel artificial maturation. Our results also implied that the neurogenesis or neural activity in the initial ovary might be an important factor influencing the outcomes of hormonal treatments. Meanwhile, more experiments and data are required to support and verify this hypothesis.

### **4. From ePAV to phenotypes**

The term "pan-genome" is the entirety of a species gene repertoire, it is defined as the complete gene set across strains rather than in a single individual. The concept of pan-genome is built upon the observation that genes often display presence-absence variations (PAV) [36]. Pan-genome comprises of the "core" genes share by all strains within a given species, "dispensable" or "accessory" genes are only present in some strains, "unique" genes are strain-specific [36]. PAV is an important source of genetic divergence and diversity [37] and has been suggested to contribute to phenotypic variation of agronomic importance in various crops [38]. With the completion of reference genome sequencing, our knowledge of genomic variation increased, it is apparent that a single reference sequence is insufficient to represent the extent of genomic variation, resulting in the introduction and growth of the pan-genome concept [39]. In nowadays, PAV has been commonly observed across the Tree of Life [39].

The pan-transcriptome can be defined by recalling the concept of the pan-genome. It reflects the set of all the RNA present in a specie or in a single organism, transcripts present in every individual of a taxonomic group are called core genes, while transcripts absent in at least a single individual are called accessory (dispensable) genes [40].

Indeed, a larger part of the genes are expression presence-absence variation (ePAV) or are even totally absent (genomic PAV; gPAV) [41]. PAV at the genomic level would be reflected in the transcriptome ePAV. The ePAV not only reflect genomic structural variation, but also the variations in genetic and epigenetic regulatory elements [41].

As a correlation between the initial ovarian status and the phenotypic outcomes of 17MT treatment have been demonstrated [5], and the genetic background of initial ovary seems important [23]. We ameliorated our results that the ovarian transcriptome was re-mapped against the Japanese genomic scaffolds, a pair as the unit to re-analyze DEGs. Certain genes were absent in one pair but present in the others, the presence genes were also up- or down-regulated, the genes of various sets were analyzed for KEGG pathway enrichment. Together up, we speculated the existence of presence-absence variant (PAV) or expression presence-absence variation (ePAV) in Japanese eel.

The 22199 pan-ovarian transcripts were derived from ovarian tissues of 12 eels with different maturation stages. In a previous report, the number of total trinity "genes" was 336,973 [23], while there were 22,199 pan-ovarian transcripts after re-mapped against the Japanese genomic scaffolds. This significant discrepancy shows the importance of specific genomic scaffolds on transcriptomics. The number of functional genes was countered under the criterion that a gene was detected in at least one of six samples (three pairs), and the gene was eliminated when this gene was not detected in any of the six samples. The number of core genes was 15466; there were 980, 501, and 1097 accessory genes for the intersection between 1440s and 2003s, between 2003s and 2613 s, and between 2613 s and 1440s, respectively. The numbers of unique genes were 1335, 796, and 724 for the 1440s, 2003s, and 2613 s, respectively (**Figure 5**). we emphasize PAVs here. We cannot confirm that the PAVs were stagespecific or inducible and would be detectable (expressed) in the later stages, since the ovaries of this stage remained far from maturation.

Regarding the phenotype, there was no significant difference in mean ovarian diameter among the initial ovarian tissues (**Figure 1**) [5], but all genotype data from both PCA and heatmaps indicate a closer distance between 1440MT (the initial ovarian tissue) and 2613MT, while 2003MT was more isolated from others (**Figures 3** and **4**) [23],

### **Figure 5.**

*Number of functional genes in each pair. Functional genes were detected in at least one sample; if not, this gene was eliminated (MT* ≠ *0* ∪ *AT*≠*0).*

### *Stimulatory Effects of Androgens on Eel Primary Ovarian Development - from Phenotypes… DOI: http://dx.doi.org/10.5772/intechopen.99582*

these observations imply that there were genetic variations. Accordingly, we speculate that the expression of the absent genes may be up-regulated (from zero to detectable) by the treatment or in the following stage; if so, these absent genes are not presence-absence variations. It has been demonstrated that, not all absent genes were developmental- or stage-specific, which indirectly supports the concept of ePAV in Japanese eel [42].

It is essential to characterize the ePAV genes and their possible functions [41], the core genes has been speculated to appear universally over-represented by housekeeping functions and essential metabolic processes for organisms, and accessory genes are often associated with communication, virulence, and defense responses ([39], references therein] or adaptive functions in plants (e.g., stress responses [43]). It has also been demonstrated that accessory genes exhibit higher rates of polymorphism than core genes [44, 45]. We studied possible functions of the unique genes of PAVs in each pair by KEGG pathway enrichment testing, since phenotypes are determined or controlled by constellations of pathways. The list of unique presence genes of each pair was submitted to Molas (http://molas.iis.sinica.edu.tw/jpeel/) to test for the KEGG pathway enrichment, and the pathways were marked when the enrichment p-value <0.05; these pathways were clustered into seven categories in the KEGG pathway database. Indeed, only approximately 36% of unique genes were hit in the KEGG pathways (the hit rates were 37%, 36%, and 36% for 1440s, 2003s and 2613 s, respectively). These results indicate that the functions of transcripts in eels remained largely unknown and non-annotated, while the results also indicated that 1440s was clearly more similar to 2003.

All signaling pathways belong to the category of "Environmental Information Processing" in KEGG, and the numbers of pathways of each pair in this category were 12, 7, and 5 for 1440s, 2003s and 2613, respectively. There were 3 pathways at the intersection of those 3 pairs: Rap1 signaling pathway, cGMP-PKG signaling pathway, and cAMP signaling pathway. This result implies the importance of these signaling pathways in basal ovarian development, and the importance of differential pathways in ovarian development was shown. Moreover, we believe that the differentially expressed genes are integrated into certain pathways to determine or control phenotypes. It has been demonstrated that the functions of genes related to neuronal activities or neurogenesis appears important in the outcomes of androgen treatments [23]. This postulation is supported the existing of ePAV in Japanese eel.

### **5. Conclusion**

Our results, from Japanese eel, implied that the initial ovary status might be an important factor influencing the outcomes of androgen treatment, and the genes related with neuronal activities or neurogenesis seemed to play an essential role in the positive effect; certain genes seemed absent in one individual but present in the others by speculating that there is a presence-absence variant (PAV) or expression presenceabsence variation (ePAV) in Japanese eel, a primitive fish diverged from other bony fishes in the basal lineage of the Teleost. Most pathways involved by ePAV were belong the endocrine system and nervous system. These results signify the importance of genetic differential expression on the variations of phenotypes by androgen, and a transcriptomic approach appears to enable extracting multiple layers of genomic data. More elegant experimental designs fit the biological model, and more transcriptomic data are required to address this question in the future.

### **Acknowledgements**

We would like to thank Mr. Yung-Lin Huang and Mr. Yi-Sen Lai, their encouragements were appreciated sincerely. Mr. Guan-Ru Chen (Freshwater Aquaculture Research Center, Fisheries Research Institute, C.O.A., Taiwan) is also appreciated for his help on the manipulation. This research was partly funded by the Ministry of Science and Technology (MOST107-2321-B002-057), Taiwan.

### **Funding**

This study was partly funded by the Ministry of Science and Technology (MOST107–2321-B002–057), Taiwan.

### **Conflict of interest**

The authors declare that they have no conflict of interest.

### **Code availability**

All software application employed are online open access.

### **Author details**

Yung-Sen Huang1 \* and Chung-Yen Lin<sup>2</sup>

1 Department of Life Science, National University of Kaohsiung, Kaohsiung, Taiwan

2 Institute of Information Science, Academia Sinica, Taipei, Taiwan

\*Address all correspondence to: yshuang@nuk.edu.tw

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Stimulatory Effects of Androgens on Eel Primary Ovarian Development - from Phenotypes… DOI: http://dx.doi.org/10.5772/intechopen.99582*

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### **Chapter 8**

## Intraovarian Gestation in Viviparous Teleosts: Unique Type of Gestation among Vertebrates

*Mari-Carmen Uribe, Gabino De la Rosa-Cruz, Adriana García-Alarcón and Juan Carlos Campuzano-Caballero*

### **Abstract**

The intraovarian gestation, occurring in teleosts, makes this type of reproduction a such complex and unique condition among vertebrates. This type of gestation of teleosts is expressed in special morphological and physiological characteristic where occurs the viviparity and it is an essential component in the analysis of the evolutionary process of viviparity in vertebrates. In viviparous teleosts, during embryogenesis, there are not development of Müllerian ducts, which form the oviducts in the rest of vertebrates, as a result, exclusively in teleosts, there are not oviducts and the caudal region of the ovary, the gonoduct, connects the ovary to the exterior. The lack of oviducts defines that the embryos develop into the ovary, as intraovarian gestation. The ovary forms the oocytes which may develop different type of oogenesis, according with the storage of diverse amount of yolk, variation observed corresponding to the species. The viviparous gestation is characterized by the possible intimate contact between maternal and embryonic tissues, process that permits their metabolic interchanges. So, the nutrients obtained by the embryos could be deposited in the oocyte before fertilization, contained in the yolk (lecithotrophy), and may be completed during gestation by additional provisioning from maternal tissues to the embryo (matrotrophy). Then, essential requirements for viviparity in poeciliids and goodeids are characterized by: **a)** the diversification of oogenesis, with the deposition of different amount of yolk in the oocyte; **b)** the insemination, by the transfer of sperm to the female gonoduct and their transportation from the gonoduct to the germinal region of the ovary where the follicles develop; **c)** the intrafollicular fertilization; **d)** the intraovarian gestation with the development of embryos in intrafollicular gestation (as in poeciliids), or intraluminal gestation (as in goodeids); and, **e)** the origin of embryonic nutrition may be by lecithotrophy and matrotrophy. The focus of this revision compares the general and specific structural characteristics of the viviparity occurring into the intraovarian gestation in teleosts, defining this reproductive strategy, illustrated in this review with histological material in a poeciliid, of the species *Poecilia latipinna* (Lesueur, 1821) (Poeciliidae), and in a goodeid, of the species *Xenotoca eiseni* (Rutter, 1896) (Goodeidae).

**Keywords:** oogenesis, gonoduct, intrafollicular gestation, intraluminal gestation, lecithotrophy, matrotrophy

### **1. Introduction**

Viviparity, the marvelous and complex reproductive strategy in which the mother retains developing eggs inside their body and give birth to a living young, occurs in all the Classes of vertebrates, except in birds. Viviparity among vertebrates makes the evolutionary first appearance in fishes, becoming essential in the understanding of this reproductive strategy [1–4]. The viviparity develops an ample diversity of features along the evolution of this type of reproduction in vertebrates, this is the case of teleosts of the families Poeciliidae and Goodeidae. The adaptations for viviparity in these species developed strategies that have been successful in a wide variety of aquatic environments [3, 5, 6]. In teleosts, during embryogenesis, there are not development of Müllerian ducts, which form the oviducts in the rest of vertebrates, as a result, exclusively in these fishes, there are not oviducts, consequently there is not uterus. Therefore, the posterior zone of the ovary of teleosts, called gonoduct, characterized by the absence of germinal cells, connects the rest of the ovary to the exterior, opening at the genital pore. The lumen of the germinal region of the ovary is continuous with the lumen of the gonoduct [1–3, 7–10]. Because of the lack of oviducts in viviparous teleosts, the development of the embryos occurs into de ovary, as an intraovarian gestation, unique in vertebrates, instead of into the uterus as occurs in the rest of viviparous vertebrates. This ovarian structure defines the characteristics of the teleosts viviparity as occurs in poeciliids and goodeids. The characteristics of the ovary during non-gestation and gestation stages are presented and compared in this study, taking as models two species: the poeciliid *Poecilia latipinna* (Lesueur, 1821), and the goodeid *Xenotoca eiseni* (Rutter, 1896). During non-gestation, ovaries during previtellogenesis and vitellogenesis where selected; and, during gestation, ovaries in early, middle, and advanced gestation where selected. The ovaries were prepared for histological analysis, stained with Hematoxiline-Eosine and the image 3A is PAS [11]. Digital photomicrographs were taken using an Olympus digital camera (model C5050Z) coupled to an Olympus CX31 microscope.

The gonadal differentiation includes the initial development of two ovaries which fuse forming a single and saccular ovary with a central lumen [1, 2]. The lack of Müllerian ducts determines the lack of oviducal development. Consequently, the female reproductive system is formed only by the ovary where the gonoduct, a non-germinal caudal portion of the ovary, connects it with the exterior [8–10]. In agreement with these structural characteristics, gestation occurs in the ovary. The embryonic development in poeciliid is intrafollicular therefore, there is not ovulation, and in goodeids is intraluminal, being the embryos discharged from the follicle to the ovarian lumen just after fertilization.

Poeciliids produce eggs generally larger than goodeids. The eggs of the most of poeciliid species attains approximately 2.0–2.5 mm in diameter, meanwhile the eggs of the most of goodeid species attains approximately 0.5–1.0 mm in diameter. Similar data for species of both families are related to time of gestation period, approximately 45–60 days of gestation, and the total length of embryos at birth about 13-20 mm total length. Then, the larger eggs of poeciliid eggs compared with smaller eggs of goodeid eggs, but similar size in the newborns indicates the more intense lecithotrophy in poeciliids compared with more intense matrotrophy in goodeids. [1–3, 6].

Consequently, goodeid embryos required to absorb more nutrients derived from maternal tissues during gestation.

### **2. Structure of the ovary in poeciliids and goodeids**

As in most viviparous teleosts, in all poeciliids and goodeids, such as *P. latipinna*, and *X. eiseni*, the ovary is a single organ, longitudinally situated. The single ovary presents a saccular structure, with a central lumen, corresponding to the cystovarian type. The single ovary is the result of the fusion of both ovaries during embryological development [2, 3, 12, 13]. The ovary is surrounded by the coelom. The ovary is located dorsally to the digestive tract and remains attached to the dorsal wall of the body by the mesovarium. The ovarian wall contains four tissue layers, they are: **a)** the germinal epithelium which lines the ovarian lumen; **b)** the stroma, subjacent to the epithelium, formed by loose vascularized connective tissue, containing follicles in different stages of development; **c)** smooth muscle layers in circular and longitudinal disposition; and **d)** serosa, formed by scarce connective tissue and mesothelium at the periphery [7, 13–17]. The ovarian mucosa forms irregular folds, that extend into the ovarian lumen, called lamellae. The lamellae are lined by germinal epithelium, where follicles in diverse stages of oogenesis may be located. The germinal epithelium contains oogonia, situated among somatic epithelial cells [3, 13, 17, 18]. The internal position of the germinal epithelium in the saccular ovary defines that the ovulation in oviparous teleosts occurs into the lumen, instead of into the coelom as happens in all other vertebrates [2, 3, 13, 16, 19].

The ovary of *P. latipinna*, and *X. eiseni*, is essentially characterized by the occurrence of the oogenesis, complex and vital process of the female germinal cells, as in all animals, during their development to mature oocytes [20]. The oogenesis in viviparous teleosts, is similar to that described in oviparous teleosts, involving cyclical sequence of morphophysiological changes in the differentiation of oogonia into full-grown oocytes. According to the annual cycles, the ovary may contain only early stages of oogenesis (previtellogenesis) (**Figure 1A** and **2A**), or late stages of oogenesis (vitellogenesis) (**Figure 1B** and **2B**). Several authors analyze this process [7, 14–16, 21–24]. Oogonia is the earliest stage of germ cells which grow from an initial diameter of approximately 10 μm. Oogonia initiate meiosis becoming oocytes which are surrounded by follicular cells, developing the primordial follicles. This process comprises two main stages in sequence: previtellogenesis (**Figure 1A**, **C**, **D** and **2A**, **C**, **D**), and vitellogenesis (**Figure 3A**–**D** and **4A**–**D**). During previtellogenesis, the nucleus of the oocyte, call germinal vesicle, has a nucleolus which proliferates to multiple nucleoli, and the ooplasm initiates the growths involving a great increase in the number of organelles associated with synthetic activities, as ribosomes, endoplasmic reticulum, mitochondria, and Golgi apparatus, becoming basophilic for the staining affinity of the ooplasm. Posteriorly, during the vitellogenesis, the oocyte growth considerably when accumulates lipid globules and yolk, becoming acidophilic for the staining affinity of the yolk. Yolk is the fundamental and more abundant material stored during oogenesis for the metabolic activities required during the embryonic development. In addition to yolk, glycogen granules and lipid globules are also stored in the oocyte, forming an essential complex for embryonic nutrition. In the full-grown oocyte, the germinal vesicle moves towards the animal pole. In both, goodeids and poeciliids, during oocyte maturation the hydration of yolk occurs and becomes fluid and homogeneous, and some lipid globules may remain around the oocyte periphery [5, 7, 13].

### **Figure 1.**

*Ovarian structure of* Poecilia latipinna*, comparing ovaries during non-gestation with previtellogenic oocytes (A) and vitellogenic oocytes (B). A) Saccular ovary with a central lumen. Ovary during non-gestation with numerous follicles during different stages of previtellogenesis seen by the change in the oocyte diameter. In some oocytes, the nucleus (germinal vesicle) is seen. The follicular cells surround the oocytes. In the caudal region of the ovary the initial portion of the gonoduct is seen. B) Saccular ovary with a central lumen. Ovary during non-gestation with follicles during late vitellogenesis containing abundant yolk fluid and homogeneous. Lipid globules are seen at the periphery. The follicular cells surround the oocytes. C) Follicles during different stages of previtellogenesis, follicular cells surround the oocytes, a group of oogonia is seen. The lumen is surrounded by the germinal epithelium. D) Two follicles in advanced stage of previtellogenesis, the basophilia diminishes with the growth of the oocyte. (fe) follicular epithelium, (ge) germinal epithelium, (gv) germinal vesicle, (go) gonoduct, (L) ovarian lumen, (lg) lipid globules, (Og) oogonia, (Oo) oocytes, (Y) yolk. Staining technique: A-D: Hematoxiline-Eosine.*

*Intraovarian Gestation in Viviparous Teleosts: Unique Type of Gestation among Vertebrates DOI: http://dx.doi.org/10.5772/intechopen.100267*

#### **Figure 2.**

*Ovarian structure of* Xenotoca eiseni*, comparing ovaries during non-gestation with previtellogenic oocytes (A) and vitellogenic oocytes (B). A) Saccular ovary with a central lumen. Ovary during non-gestation with follicles during different stages of previtellogenesis seen by the change in the oocyte diameter. B) Saccular ovary with a central lumen. Ovary during non-gestation with follicles during late vitellogenesis containing abundant yolk. In some oocytes, the nucleus (germinal vesicle) is seen. The follicular cells surround the oocytes. C) Follicles with oocytes in different stages of previtellogenesis, follicular cells surround the oocytes. The lumen is surrounded by the germinal epithelium. D) Detail of oocytes in early stages of previtellogenesis. The nucleus (germinal vesicle) is seen. The follicular cells surround the oocytes. (fe) follicular epithelium, (ge) germinal epithelium, (gv) germinal vesicle, (L) ovarian lumen, (Oo) oocytes, (Y) yolk. Staining technique: A-D: Hematoxiline-Eosine.*

### **Figure 3.**

*Ovary of* Poecilia latipinna*. A) Ovary during non-gestation with follicles during the initial vitellogenesis, with yolk platelets at the periphery. Lipid globules are seen among the yolk. The acidophilia is clear by the presence of the yolk. The ovarian lumen is surrounded by the germinal epithelium. B) Follicle in advanced stage of vitellogenesis, the yolk is initially becoming fluid and homogeneous. Abundant lipid globules are seen. C) Portion of a vitellogenic oocyte, with lipid globules at the periphery, the yolk is fluid and homogeneous. The follicular cells surround the oocytes. Compare this vitellogenic oocyte with the previtellogenic oocyte seen in the upper part of the image. D) Late vitellogenic oocyte, the yolk is completely fluid and homogeneous, some lipid globules may be seen around the oocyte periphery. (fe) follicular epithelium, (ge) germinal epithelium, (gv) germinal vesicle, (L) ovarian lumen, (lg) lipid globules, (Oo) oocytes, (Y) yolk. Staining technique: A: PAS; B-D: Hematoxiline-Eosine.*

There is a clear difference between the egg size diameter of mature eggs of poeciliids and goodeids, been larger the eggs of poeciliids. In most poeciliid species there is a mean diameter of mature eggs of 1.5 mm to 2.5 mm [6, 25–27]. Even there are some poeciliids with smaller oocytes as *Heterandria formosa*, which mature oocyte attains an egg diameter of 0.4 mm, considered the smallest egg of poeciliids [28]. The species *Intraovarian Gestation in Viviparous Teleosts: Unique Type of Gestation among Vertebrates DOI: http://dx.doi.org/10.5772/intechopen.100267*

*P. latipinna* forms mature eggs with a mean diameter of 2.2 mm [26]. In most goodeid the mature egg has a mean diameter of 0.6 mm to 1.0 mm., the species *X. eiseni* forms mature eggs with a mean diameter of about 0.8 mm in diameter [29].

### **Figure 4.**

*Ovary of* Xenotoca eiseni*. A) Ovary during non-gestation with follicles in the initial vitellogenesis, with yolk platelets at the periphery. Lipid globules are seen among the yolk. The follicular cells surround the oocytes. The acidophilia is clear by the presence of the yolk. The ovarian lumen is surrounded by the germinal epithelium. B) Follicle in middle stage of vitellogenesis, the yolk is initially becoming fluid and homogeneous. Abundant lipid globules are seen. The follicular cells surround the oocyte. C) Late vitellogenic oocyte, with lipid globules at the periphery, the yolk is fluid and homogeneous. The follicular cells surround the oocytes. The nucleus (germinal vesicle) is seen at the animal pole. Compare this vitellogenic oocyte with the previtellogenic oocytes seen in the upper part of the image. D) Late vitellogenic oocyte, the yolk is completely fluid and homogeneous, some lipid globules may be seen around the oocyte periphery. (fe) follicular epithelium, (ge) germinal epithelium, (gv) germinal vesicle, (L) ovarian lumen, (lg) lipid globules (Oo) oocytes, (Y) yolk. Staining technique: A-D: Hematoxiline-Eosine.*

### **3. Fertilization**

Viviparity is basically related to the evolution of reproductive behavior, insemination and fertilization are essential requirements for viviparity. In viviparous species, as poeciliids and goodeids, the internal fertilization involves the insemination, with the entrance of spermatozoa to the female reproductive system, through the transfer of sperm from the male to the female gonoduct, and the sequence of the entrance of the sperms, from the gonoduct to the germinal zone of the ovary, where occurs the internal fertilization [1, 19, 30–32]. The secretory activity of the ovarian epithelium, having a trophic function for the spermatozoa, contributes for their survival during the appropriate time for the fertilization. In *P. latipinna*, and *X. eiseni*, the

### **Figure 5.**

*Ovary of* Poecilia latipinna*. A) When insemination occurred, the ovary contains abundant spermatozoa in the lumen. Several previtellogenic oocytes are seen, surrounded by follicular epithelium. B) Ovary during intrafollicular gestation with embryos in early stage of development, after neural tube formation, with abundant yolk. Lipid globules are seen around the yolk. The follicular epithelium surrounds the follicle in gestation. C) with the advance of the gestation, the embryo growths and the yolk reduce. Lipid globules continue at the periphery. The follicular epithelium surrounds the follicle in gestation. (E) Embryo, (fe) follicular epithelium, (gv) germinal vesicle, (L) ovarian lumen, (lg) lipid globules, (Oo) oocytes, (Y) yolk, (Z) spermatozoa. Staining technique: A-C, Hematoxiline-Eosine.*

### *Intraovarian Gestation in Viviparous Teleosts: Unique Type of Gestation among Vertebrates DOI: http://dx.doi.org/10.5772/intechopen.100267*

spermatozoa may be seen in the ovarian lumen (**Figure 5A** and **6A**), many of them show their heads in contact with the apical end of the germinal epithelial cells.

Insemination in viviparity may allow the temporal separation of mating and fertilization, and consequently, the temporal diversification of the time between insemination and birth, by the possibility of sperm storage in the ovary. Additionally, the sperm storage permits that, with one insemination, several clutches of eggs maturing on several occasions, may be fecundated [31].

In most viviparous teleosts, as poeciliids and goodeids, fertilization occurs inside the follicle, as an intrafollicular fertilization, therefore, there is not ovulation. During the fertilization, sperm must penetrate the follicular wall to reaches the oocyte membrane [31, 33–35].

#### **Figure 6.**

*Ovary of* Xenotoca eiseni*. A) When insemination occurred, the ovary contains abundant spermatozoa in the lumen. A previtellogenic oocyte is seen. B) Ovary during intraluminal gestation with embryos in the lumen during blastula, with abundant yolk. C) Detail of an embryo during blastula in the ovarian lumen. D) Embryo in intraluminal gestation in early stage of development, after neural tube formation, with abundant yolk. (E) Embryo, (L) ovarian lumen, (Oo) oocyte, (Y) yolk, (Z) spermatozoa. Staining technique: A-D: Hematoxiline-Eosine.*

### **4. Intrafollicular and intraluminal gestation in poeciliids and goodeids**

After the intrafollicular fertilization the embryogenesis may follows two different ways of gestation: **a)** intrafollicular gestation, where the embryos remain in the follicle during their development and move into the ovarian lumen immediately before birth, as occurs in poeciliids; or **b)** intraluminal gestation, in which the embryos, during early development as blastula, move from the follicle to the ovarian lumen where gestation continues until birth, as occurs in goodeids, in this type of gestation the embryos in the lumen are surrounded by a fluid, call histotrophe, secreted by the ovarian

### **Figure 7.**

*Ovary of* Poecilia latipinna *in intrafollicular gestation. A) Ovary during gestation with an embryo in middle stage of development. Lipid globules are seen at the periphery. Around the yolk is seen the yolk sac. The follicular epithelium surrounds the follicle in gestation. Oocytes are seen in the ovarian wall. B) Periphery of the yolk, where it is seen the vascularized yolk sac with blood vessels. Lipid globules are seen. The follicular epithelium surrounds the follicle in gestation. C) an embryo during advanced stage of gestation, the amount of yolk diminishes with the growth of the embryo. Lipid globules are seen. The follicular epithelium surrounds the follicle in gestation. (bv) blood vessels, (E) embryo, (fe) follicular epithelium, (L) ovarian lumen, (lg) lipid globules, (Oo) oocytes, (Y) yolk, (Ys) yolk sac. Staining technique: A-C, Hematoxiline-Eosine.*

### *Intraovarian Gestation in Viviparous Teleosts: Unique Type of Gestation among Vertebrates DOI: http://dx.doi.org/10.5772/intechopen.100267*

epithelium to the lumen [1, 29, 30, 36, 37]. Thus, the intrafollicular gestation is the case of the poeciliid *P. latipinna*, and the intraluminal gestation is the case of the goodeid *X. eiseni*. Embryos of *P. latipinna* in early intrafollicular gestation (**Figure 5B** and **C**) and embryos of *X. eiseni*, in early intraluminal gestation (**Figure 6B**–**D**), are seen. Embryos of *P. latipinna* in late intrafollicular gestation (**Figure 7A**–**C**) and embryos of *X. eiseni*, in late intraluminal gestation (**Figure 8A**–**C**), are seen.

### **Figure 8.**

*Ovary of* Xenotoca eiseni *in intraluminal gestation. A) Ovary during gestation with embryos in the lumen during middle stage of development, with early development of eyes. Reduced yolk in the ventral side of the embryo. Oocytes are seen in the ovarian wall. B) Ovary with embryos in the lumen during advanced stage of development. There is not yolk. C) Ovary with embryos during advanced stage of gestation. The trophotaeniae is seen as extensions of the intestine to the ovarian lumen. (E) Embryo, (L) ovarian lumen, (Oo) oocytes, (T) trophotaeniae (Y) yolk. Staining technique: A-C: Hematoxiline-Eosine.*

Consequently, both types of gestation, intrafollicular and intraluminal, define different relationships between the tissues of the embryos and the mother and successively, diverse adaptations for metabolic interchanges between them, reflected mainly in the type of embryonic nutrition. Nutrition during gestation in viviparous species reflects essential strategies which impact in the evolution of vertebrate viviparity, as occurs in goodeids and poeciliids [19, 38, 39].

In recognition of vertebrate diversity, we follow Blackburn & Starck [40] in using the term "embryo" in the broad sense, to include all types of developing offspring in gestation.

### **5. Embryonic nutrition by lecithotrophy and matrotrophy**

The maternal–embryonic relationships include trophic, osmoregulatory, excretory, respiratory, endocrinological, and immunological processes [4, 36, 37, 40]. These interchanges are essentially related to the diversity of mechanisms of embryonic nutrition. During intraovarian gestation of poeciliids and goodeids, two types of embryonic nutrition may occur: **a)** lecithotrophy, wherein the nutrients are provided by the yolk stored in the oocyte during oogenesis; and, **b)** matrotrophy, where the nutrients are transferred by maternal tissues to the embryo during gestation. Both types of nutrition are not mutually excluding, may occurs both with different degree of nutrient transfer. These strategies require a modification in the timing of maternal resource offered to the embryo, these are: during oogenesis, previously to fertilization, in lecithotrophy, and posteriorly to fertilization, in matrotrophy. Several studies analyze the adaptations from lecithotrophy to matrotrophy [4, 19, 27, 29, 38, 40–44].

In lecithotrophic species, the yolk develops a big and vascularized yolk sac around the yolk, in the ventral part of the embryo (**Figure 7A** and **B**). The yolk sac has a simple epithelium and a plexus with abundant capillaries, structures that allow the reception of the nutrients from the yolk and their distribution to the rest of the embryonic body, throughout the circulation. During gestation, the yolk sac progressively diminishes (**Figure 7C**), as the yolk is used for the nutrition of the embryo [19, 45, 46].

The viviparity generates the opportunity for the embryo to acquire resources from the mother, developing specialized structures for interchanges, as it is the development of placental structures [27, 42, 47]. In the intrafollicular gestation, maternal tissues (follicular epithelium and the subjacent net of capillaries), and embryonic structures establish the maternal–embryonic relationships during gestation [3, 19].

According to the differences of amount of yolk, the species having eggs with abundant yolk are lecithotrophic, even some transfer of maternal nutrients may also occur through the contact of the maternal blood vessels subjacent to the follicular epithelium, which surrounds the embryo in development, as occurs in poeciliids, as *P. latipinna* (**Figure 5B** and **C**). In contrast, the species having eggs with scarce yolk deposited during oogenesis, observed during early gestation in goodeids as *X. eiseni* (**Figure 6B**–**D** and **8A**), but, when the scarce yolk is early consumed, and the embryo needs nutrients from the mother for the continuation of the development are matrotrophic (**Figure 8B** and **C**).

Lecithotrophy has been revealed to be better adapted energetically to seasonally unpredictable habitats, whereas matrotrophy requires a predictable food supply during gestation. Therefore, matrotrophy requires stable conditions throughout gestation for a constant transfer of nutrients from the mother to the embryos. Then, the condition of the habitat determines the convenience of lecithotrophy or matrotrophy [42, 47–49].

### *Intraovarian Gestation in Viviparous Teleosts: Unique Type of Gestation among Vertebrates DOI: http://dx.doi.org/10.5772/intechopen.100267*

Early in the embryogenesis of goodeids, when the yolk is completely consumed (**Figure 8B** and **C**), the embryonic growth is progressively dependent of maternal nutrients through matrotrophy, initially by the nutrients dissolved in the histotrophe and then, by the transfer of nutrients via the development of complex and diverse placental structures where occurs the apposition of embryonic and maternal tissues [29, 36, 39, 43, 50–52]. Several embryonic structures have been considered for the transfer of nutrients from the histotrophe such as: the skin, the gills, the digestive tract through the mouth, and the characteristic structure of goodeids, the trophotaeniae [1, 7, 29, 39, 52–54]. The trophotaeniae is seen as extensions of the intestine to the ovarian lumen (**Figure 8C**). Additionally, transfer of nutrients occurs by placental structures, such as: the apposition of trophotaeniae to maternal epithelium, or by branquial placenta, a structure formed when folds of ovarian tissue enter the embryonic branchial chamber developing apposition of the ovarian folds to the embryonic branquial epithelium [49, 52, 55].

Embryos developing in the ovarian lumen, as *X. eiseni* (**Figure 6C**, **D** and **8A**–**C**), are bathed by histotrophe, a fluid rich in nutrients containing abundant proteins derived from the maternal blood, secreted by the ovarian tissues, and discharged in the lumen [29, 55]. The absorption of these nutrients is reached by goodeid embryos through long and vascularized extensions of the hindgut that grow outside of the embryo into the ovarian lumen developing extraordinary absorptive structures call trophotaeniae (**Figure 8C**). Trophotaeniae is also involved in gas exchange and develop process of immunity [19, 29, 30]. This capacity of absorption is evidenced by the morphological features of the trophotaeniae, as the surrounding columnar epithelium with apical brush-border and the abundant vascularization subjacent to the epithelium [29]. Then through the epithelium, the blood vessels of the trophotaeniae transport the contains from the histotrophe to the embryo. Numerous morphological and experimental studies of the trophotaeniae analyzed the transfer of nutrient to the embryonic body, as: [29, 39, 50, 52–54]. Ecdysis of the trophotaeniae occurs at birth; when the postnatal phase initiates the oral nutrition [19, 30, 36, 53].

In poeciliids during birth, the embryos are going from the follicle to the ovarian lumen and then through the gonoduct to the exterior. In goodeids during birth, the embryos are already in the ovarian lumen, then through the gonoduct they move to the exterior.

### **6. Conclusions**

The structure of the ovary and the lack of oviducts of teleosts define the characteristics of the viviparity with the intraovarian gestation, as it was presented morphologically in the sequence of Figures of ovarian histology of the poeciliid *P. latipinna*, and the goodeid *X. eiseni* in this analysis.

The ovary of viviparous teleosts is not only the organ where occurs the oogenesis, but also it receives the spermatozoa during insemination, occurs the fertilization of oocytes and the embryos remain throughout their development until birth, making the intraovarian gestation of viviparous teleosts a such complex and unique type of gestation in vertebrates.

The oogenesis is similar to that described in oviparous teleosts, since the formation of the primordial follicle, integrated by the oocyte surrounded by the follicular epithelium, and through previtellogenesis and vitellogenesis. In accordance with the species, as it is seen in poeciliids and goodeids, the oogenesis organizes oocytes with different diameters corresponding to the amount of yolk.

The intraovarian gestation may be as: **a)** intrafollicular, where the embryos remain in the follicle during their development and move into the ovarian lumen immediately before birth, or **b)** intraluminal, in which the embryos, during early development move from the follicle to the ovarian lumen where gestation continues until birth.

The origin of embryonic nutrition occurs by lecithotrophy and matrotrophy developing active structures in the transfer of nutrients to the blood vessels of the embryo. The lecithotrophy, occurring in the intrafollicular gestation of *P. latipinna*, develops the yolk sac, which decrease progressively during gestation. The matrotrophy occurring in the intraluminal gestation of *X. eiseni,* allows the provision of nutrients from the histotrophe, and develop the trophotaeniae and the branchial placenta.

### **Acknowledgements**

The authors are grateful to Harry J. Grier (In Memoriam) for valuable insightful reflections about this theme.

### **Author details**

Mari-Carmen Uribe\*, Gabino De la Rosa-Cruz, Adriana García-Alarcón and Juan Carlos Campuzano-Caballero Facultad de Ciencias, Laboratorio de Biología de la Reproducción Animal, Departamento de Biología Comparada, Universidad Nacional Autónoma de México, Ciudad de México, México

\*Address all correspondence to: mari3uribe3@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Intraovarian Gestation in Viviparous Teleosts: Unique Type of Gestation among Vertebrates DOI: http://dx.doi.org/10.5772/intechopen.100267*

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### *Edited by Yusuf Bozkurt and Mustafa Numan Bucak*

Reproduction is the backbone of animal-based food production. The reproductive systems of animals vary and are species-dependent. In this regard, all terrestrial animals perform internal fertilization, whereas aquatic animals perform different reproductive strategies such as internal fertilization without mating, external fertilization, viviparous, oviparous, and parthenogenesis. Today, reproductive biotechnology is an important part of the conservation and propagation of animals. This book addresses several hot topics in the field of reproduction of terrestrial and aquatic animals. Over five sections and eight chapters, this volume examines subjects such as cryopreservation, embryo transfer, avian reproduction, intraovarian gestation, and more.

### *Rita Payan Carreira, Veterinary Medicine and Science Series Editor*

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