Spermatogenesis and sperm transit through the epididymis in mammals with emphasis on pigs. Theriogenology, 63 2 , Previous Next. Anatomy The reproductive tract of the boar consists of different structures: Testicular and epididymis anatomy We will detail a little more these two parts since in them sperm are produced and mature, so their knowledge will help us to understand possible alterations of sperm quality that we see in the laboratory. Picture 1: B oar reproductive tract.
Inside the tubules, sperm cells will be formed, these tubules are covered by a seminiferous epithelium formed mainly by: Sertoli cells play a fundamental role in testicular development as they nourish and protect germ cells.
These cells during fetal development are responsible for sexual differentiation and, as we will see later, in spermatogenesis.
The proliferation of these cells occurs in two waves the first over the first month of life and the second at months. FSH produced in the pituitary gland has a direct effect on the maturation of these cells. Sertoli cells stop dividing before puberty regulated by thyroid hormone T3 and their number will determine future sperm production, since without the structural and metabolic support of these cells, the germ cells would not be able to perform spermatogenesis.
For this reason, the measurement of testicular size in the pre-pubertal stage is so important when selecting boars for production. Leydig cells, or steroididogenic cells , have the ability to synthesize and secrete steroids, enzymes, and peptides. These cells are located in the interstitium around the blood vessels so their hormonal secretion easily passes into the circulation.
These cells provide support and are responsible for the production of testosterone. In contrast to Sertoli cells, more than the size of them, the capacity to produce testosterone is linked to the amount of smooth endoplasmic reticulum present in them. Furthermore, they are responsible for sexual differentiation, testicular growth and the appearance of secondary male sexual characters and androgen-dependent structures and spermatogenesis in the boar.
Germ cells : the proliferation of these cells during the first weeks after birth is responsible for the formation of seminiferous tubules. The number of germ cells in the testicle increases continuously, with a peak of division at months and a stabilization of the population after 7 months. These cells are responsible for carrying out spermatogenesis, through a series of mitotic missions, followed by a series of meiotic divisions ending with the result of a haploid cell.
Picture 2: Seminiferous tubule histological picture. Source: University of Zaragoza. Picture 3: Seminiferous tubule histological picture. Histologically along the duct, we can differentiate four cell types: Main or columnar cells, responsible for the transport, secretion, synthesis and absorption of molecules.
Basal cells, metabolic waste disposal. Clear Cells, remove any material or residue present in the lumen. Halo cells, belonging to the immune system. These glands are: Seminal vesicles: they are two glands, joined together, located dorso-lateral to the neck of the bladder, and they flow into the urethra through a proper duct.
Its secretion is part of the post-sperm fraction, where the number of sperm is lower. They produce a fructose-rich alkaline secretion that protects the sperm from abrupt changes in pH and gives volume to the ejaculate. Prostate : odd and smaller gland in the boar. It secretes a liquid rich in amino acids, citric acid and enzymes.
The function of these components is to stimulate the movement of sperm. Artifacts were rarely seen and were not included in the data. Points were classified as one of the following: seminiferous tubule comprising tunica propria, epithelium, and lumen , Leydig cell, connective tissue, blood and lymphatic vessels. The volume of each testis component was determined as the product of the volume density and the testis volume.
For subsequent morphometric calculations, the specific gravity of testis tissue was considered to be 1. Stages of the cycle in wild boars were characterized based on the shape and location of spermatid nuclei, the presence of meiotic divisions, and the overall seminiferous epithelium composition.
This method, known as the tubular morphology system [ 7 ], provides eight stages of the seminiferous epithelium cycle. The duration of each spermatogenic cycle was estimated based on the stage frequencies and the most advanced germ cell type labeled at different periods investigated after thymidine injections. The total duration of spermatogenesis comprised approximately 4. All germ cell nuclei and SC nucleoli present at stage 1 were counted in 10 round or nearly round seminiferous tubule cross-sections chosen randomly for each animal.
In addition, newly formed round spermatids present at stage 5 were counted in 10 seminiferous tubule cross-sections. These counts were corrected for section thickness and for nucleus or nucleolus diameter according to the method by Abercrombie [ 23 ], as modified by Amann and Almquist [ 24 ]. For this purpose, 10 nuclei or nucleoli diameters were measured for each cell type analyzed per animal. Cell ratios were obtained from the corrected counts obtained at stage 1.
The total number of SCs was determined from the corrected counts of SC nucleoli per seminiferous tubule cross-section, and the total length of seminiferous tubules as described by Hochereau-de Reviers and Lincoln [ 25 ].
The individual Leydig cell volume was obtained based on the nucleus volume and on the proportion between the nucleus and cytoplasm. For each animal, points over Leydig cells were counted. Because the Leydig cell nucleus in wild boars is spherical, its nucleus volume was obtained based on the mean nuclear diameter. For this purpose, the diameters of 30 nuclei showing evident nucleolus were measured for each animal.
The number of Leydig cells per testis was estimated from the individual Leydig cell volume and from the volume occupied by Leydig cells in the testis parenchyma. The mean testis weight in wild boars was approximately 64 g, with a gonadosomatic index GSI testes mass divided by body weight of approximately 0.
Based on the volumes of the testis parenchyma testis weight minus [tunica albuginea plus mediastinum weight] and the seminiferous tubules and on the mean tubular diameter, the mean seminiferous tubular length per gram of testis was 18 m, and the mean total seminiferous tubular length per testis was m Table 1.
The eight stages of the seminiferous epithelium cycle in wild boars are briefly described in the legend to Figure 1. Stage 6 had the highest frequency, and stages 1 and 8 had the lowest frequencies. The premeiotic stages 1—3 and postmeiotic stage 5—8 stages occupied approximately one third and one half of the total stage frequencies, respectively. Stages 1 to 8 of the seminiferous epithelium cycle based on the tubular morphology system.
The most advanced germ cell types labeled at different periods investigated after thymidine injections are given in Table 2 and in Figure 2. At approximately 1 h after injection, the most advanced germ cell types labeled were preleptotene spermatocytes or cells in transition from preleptotene to leptotene.
These cells were present at the end of stage 2 and were located in the basal compartment Fig. The most advanced germ cell types labeled at 7 days after thymidine injection were pachytene spermatocytes, present at the end of stage 8 Fig. Labeled round spermatids at stage 6 were observed at 14 days after thymidine injection Fig. The most advanced germ cell types labeled after different periods following intratesticular injections of tritiated thymidine were as follows: a At 1 h after injection, leptotene primary spermatocytes arrows at the end of stage 2.
The duration of the various stages of the cycle according to the tubular morphology system was determined, taking into account the cycle length and the percentage of occurrence of each stage Fig. The shortest duration 0. Considering that approximately 4. The duration of the spermatogonial phase of spermatogenesis was approximately 14 days, and the life spans of primary spermatocytes and spermatids were approximately 13 days and 14 days, respectively.
Diagram showing the germ cell composition, frequencies percentage , and duration in days for each stage of the seminiferous epithelium cycle. Also depicted are the most advanced germ cell types labeled at the eight stages of the cycle at the different periods investigated at 1 h and at 7, 14, and 21 days following tritiated thymidine injections.
Roman numerals indicate the spermatogenic cycle. The space given to each stage is proportional to its frequency and duration. The letters within each column indicate germ cell types present at each stage of the cycle. A indicates type A spermatogonia; In, intermediate spermatogonia; B, type B spermatogonia; Pl, preleptotene spermatocytes; L, leptotene; Z, zygotene; P, pachytene; D, diplotene; II, secondary spermatocytes; R, round spermatids; E, elongated spermatids.
The mean numbers of Leydig cells and SCs per testis were approximately 10 billion and 2. The SC efficiency in wild boars, estimated from the total number of round spermatids per SC, was approximately 7. These data show that almost one third of the germ cells were lost during the two meiotic divisions in the wild boar seminiferous epithelium. The DSP values per testis and per gram of testis among wild boars in this study were 1.
To our knowledge, the present study is the first to report the results of a careful morphofunctional investigation of the testis structure and the spermatogenic process in the wild boar. Besides providing valuable data regarding the reproductive biology of this species, our results may allow important comparisons between the well-investigated domestic pig and the wild boar, which is phylogenetically the ancestor of the domestic pig.
Although wild boars in their natural European habitat show seasonal reproduction, as observed for ovarian and testis activity [ 27 — 30 ], reproduction in this species in the tropical climate of Brazil seems unaffected by season, as offspring are born throughout the year.
For this reason, we did not investigate the influence of season on testis function. Despite this age difference, the GSIs obtained in both studies were similar. This may be explained by the extensive selection for reproductive performance to which the domestic pig breeds have been submitted. Table 4 compares several important testicular parameters between domestic pigs and wild boars. Comparative parameters related to the testis morphometry and spermatogenic events in sexually mature domestic and wild boars.
These data are related to sexually mature boars and from several different pig breeds such as Yorkshire, Lacombe, Landrace, Whitecross, Piau, Meishan, and West African. One of the main causes of different spermatogenic efficiencies observed among mammalian species seems to be the variation in the proportion of tubular and intertubular compartments [ 12 , 17 , 32 ].
The mean percentage of the intertubular compartment in the wild boar was approximately two thirds of the reported value for the domestic pig [ 12 , 31 , 33 ]. Because Leydig cells secrete steroids and pheromones that are important for other reproductive functions in boars such as sexual behavior and maintenance of the male reproductive tract function and accessory glands , the domestic pig may have required a higher volume density of Leydig cells during the selective process for high reproductive efficiency.
However, the number of Leydig cells per gram of testis observed for wild boars is considerably higher that the values cited for domestic pigs [ 12 , 33 ] and is the highest found among mammalian species investigated to date [ 12 , 34 ].
On the other hand, the individual Leydig cell size in wild boars was approximately 5-fold smaller than the mean size observed for domestic pigs [ 33 ] and represents one of the smallest Leydig cells observed among mammals [ 12 , 34 ]. To our knowledge, there is no explanation in the literature about what determines the Leydig cell cytoarchitecture organization in the testis and its volume density and individual size.
Probably because of the phylogenetic proximity between wild boars and domestic pigs, the germ cell morphology and the different cellular associations characteristic of each stage of the seminiferous epithelium cycle were similar in these two swine subspecies [ 17 , 35 , 36 ]. However, the same was not true for the stage frequencies. Reports showing high variation of the stage frequencies among different individuals of the same mammalian species are not unusual in the literature [ 8 , 9 , 36 , 39 , 40 , 41 ], and this feature was observed in wild boars investigated in the present study.
However, when wild boars and domestic pigs are compared, the variation observed for the stage 3 frequency indicates that the pace of germ cells in this particular stage of spermatogenesis has changed drastically during evolution and domestication.
The reason for this is unknown, but it is probably related to changes in the cell cycle or in the germ cell complement that is characteristic of this stage. Nevertheless, when the frequencies of different stages are grouped in premeiotic and postmeiotic phases of spermatogenesis, the values were similar for wild boars and domestic pigs [ 12 , 35 , 36 , 42 ].
This is in agreement with several reports in the literature [ 12 , 36 , 40 , 43 , 44 ], suggesting that the phylogeny is strongly related to the frequencies of different stages when they are grouped in premeiotic and postmeiotic phases. The autoradiographic technique we used allowed high-quality labeling for germ cells in the studies of the duration of the spermatogenic events.
The values obtained in wild boars for the spermatogenic cycle length and the total duration of spermatogenesis were similar to those found for domestic pigs [ 35 , 36 ]. Although high variation was observed among the frequencies for some stages and, consequently, for the germ cell pace, the total duration of spermatogenesis was not different in these two swine species.
This finding is in general agreement with investigations showing that the spermatogenic cycle length is under the control of the germ cell genotype [ 15 ] and that the duration of spermatogenesis does not differ substantially among different breeds or strains [ 6 , 12 ]. Each of the three phases of spermatogenesis spermatogonial, meiotic, and spermiogenic lasts approximately one third of the entire process [ 9 , 12 , 40 ], and the values obtained for these spermatogenic phases are similar in both swine species.
In mammals, only 2 or 3 of 10 expected spermatozoa are produced from differentiated type A spermatogonia, and the highest level of germ cell apoptosis occurs during the spermatogonial phase through a density-dependent regulation [ 45 ] and during meiosis due to chromosomal damage [ 12 , 46 ]. The meiotic index observed for wild boars in the present study was similar to that found for other swine breeds [ 12 ] and is slightly higher than the values observed for most other species [ 32 , 46 ], suggesting that this aspect of spermatogenesis is conserved in pigs.
Based on the fact that each SC supports a limited number of germ cells in a species-specific manner [ 47 ], the number of SCs established before puberty determines the rate of sperm production in sexually mature animals [ 16 , 17 , 48 — 50 ]. Taking these assumptions into consideration, the spermatogenic efficiency, expressed as the DSP per gram of testis, is usually positively correlated with the number of germ cells supported by each SC [ 12 , 17 , 32 , 51 ].
The number of SCs per gram of testis for wild boars was much higher than that found in domestic swine breeds and in most other mammalian species [ 17 , 38 ]. This result suggests that the reproductive selection process in domestic pigs has improved the SC efficiency, resulting in significantly higher testis weight and GSI compared with those in wild boars. The spermatogenic efficiency is considered a useful parameter for interspecies comparisons among mammals [ 7 , 12 , 14 , 17 , 51 ].
The spermatogenic efficiency observed in wild boars is slightly higher than that found in domestic pigs [ 12 , 17 , 35 , 38 , 52 — 56 ]. As summarized in Table 4 , this indicates that the lower SC efficiency in wild boars is compensated by a higher number of SCs per gram of testis.
However, because of the much smaller testis size observed in this species, its DSP per testis is low compared with that in domestic pigs [ 53 ]. Nowak RM. Google Scholar. Google Preview. The origin of the domestic pig: independent domestication and subsequent introgression. Genetics ; : — Wild boar crosses in Argentine: growth, feed conversion and carcass. InVet ; 3 : 49 — Gimenez DL.
Berndtson WE. Methods for quantifying mammalian spermatogenesis: a review. J Anim Sci ; 44 : — Testis morphometry, seminiferous epithelium cycle length, and daily sperm production in domestic cats Felis catus.
Biol Reprod ; 68 : — The seminiferous epithelium cycle length in the black tufted-ear marmoset Callithrix penicillata is similar to humans. These genes may serve crucial roles in regulating testis development and their expression may induce sexual maturity and spermatogenesis.
Wnt signaling serves an essential role during testis development and can impact testis-related disorders Many functional genes related to spermatogenesis or reproduction in pigs involve the p53, mitogen-activated protein kinases MAPK and Wnt signaling pathways The mammalian target of rapamycin mTOR pathway was involved in regulating the proliferation of rat Sertoli cells The prostate is one of the male accessory sex organs; prostate cancer significantly influences the reproductive system and is among the most frequently diagnosed solid tumours among men Steroidal testosterone depletion retards testes growth, reduces the relative weight of the testes and accessory sex organs and reduces sperm counts and motility We also annotated the GnRH-mediated and epigenetic regulation pathways and verified their involvement in the regulation of sexual maturity development 32 , A previous study reported that in vivo , conditional and pituitary-specific disruption of ERK extracellular signal-regulated kinase signaling by GnRH caused to a gender-specific perturbation of fertility Epigenetic regulation is one of the major factors that regulate gene expression in response to environmental stimuli; this regulation involves chemical modification of DNA cytosine residues without altering the DNA sequence Aberrant DNA methylation patterns of spermatozoa in men affect fertility and sperm function Our RNA-seq data identified many epigenetic genes that were significantly differentially expressed between Duroc and Meishan boars at different ages.
We speculated that the genes also served critical roles in inducing differences in male fertility between Duroc and Meishan boars and between different ages within the breed, as DNMT3A conditional mutant males were previously demonstrated to exhibit impaired spermatogenesis Alternative splicing is one of the most important RNA modifications. This process causes protein diversification and contributes to the complexity of higher organisms 38 , This study detected four primary alternatively spliced events.
We also detected a large proportion of genes that contain one or more alternatively spliced events from the six samples see Supplementary Fig. Alternative splicing is considered to be a key factor that increases cellular and functional complexity in eukaryotes SNPs were detected in coding sequence; most of the genomic variation was attributed to SNPs, which may also increase the functional complexity and offer the highest resolution for tracking disease genes and population history Approximately 60,—, SNPs from each sample were identified based on specific matching to the Sus scrofa genome and the numbers of SNPs in the Meishan boars were greater than in the Duroc boars.
The SNP data are useful for the identification of candidate genes and biomarkers for boar fertility 15 , A larger number of SNPs may exist in the non-coding sequences compared with the coding sequences. It deserves further investigation by whole-genome sequencing. The selecting of candidate genes to explore the regulatory mechanism of puberty will be the subject of a future study. After searching for new differentially expressed transcripts and genes in QTLs related to reproduction traits as shown in Table 2b , we determined that the number of significant DEGs and novel transcripts between breeds at 75 days was substantially larger than the same number at 20 and days.
More DEGs were detected between 20 and 75 days in Meishan boars, which validated the differences in the reproductive traits between Meishan and Duroc boars. S6 online. NKX in the mature testis can cause spermatogenic cell division and differentiation and prompt spermatogenesis if it is suppressed We identified genes related to spermatogenesis and the regulation of mammalian reproduction.
Therefore, it was worthwhile to validate the expression of the genes. NTF3 was involved in the progression of male sex differentiation and was critical to the induction of embryonic testis cord formation 46 , Our study showed that NTF3 was significantly differently expressed between Duroc and Meishan boars at different ages and that its expression decreased at adulthood, which suggests that NTF3 also served an important role in the early development of testes.
An immunohistochemical assay indicated NTF3 was localized on E14 and E16 in embryonic rat testis Sertoli cells and was expressed in germ cells and Leydig cells at E16 Our study demonstrated NTF3 was primarily localized in spermatogonia and was detected at low levels in the spermatocyte and sperm; however, no expression was detected in the Sertoli cell after birth.
Therefore, we deduced that NTF3 also served a significant role in postnatal spermatogenesis. One explanation may be that post-transcriptional regulation caused the different expression patterns between the RNA and protein levels.
Histological analyses illustrated that Meishan boars underwent puberty and sexual function development at an earlier stage than Duroc boars. RNA-seq enabled us to obtain potential signature genes and discover pathways that were significantly involved in the regulation of male sexual function.
A large number of alternative splicing events and SNPs that detected in the testes require further analysis. The analyses of DEGs and QTLs related to reproduction aided in our understanding of the molecular mechanisms that control the sexual development of Duroc and Meishan boars.
This study will provide a valuable transcriptomics resource for Duroc and Meishan boars and enable researchers to identify functional genes, molecular markers and pathways that influence sexual function development and spermatogenesis.
All animals were raised under the same conditions. The methods were performed in accordance with the approved guidelines from Huazhong Agricultural University and scientific, ethical and legal principles of the Hubei Regulations for the Administration of Affairs Concerning Experimental Animals. All experimental protocols were approved by the Ethics Committee of Huazhong Agricultural University. The samples were washed with running water, dehydrated using an ethyl alcohol series, cleared in xylene and embedded in paraffin wax.
Japan biomicroscope. Significance analyses that pertain to the diameters of the seminiferous tubules were conducted using a T-test. The trimmed reads were assembled and mapped to the reference genome Sscrofa Values of RPKM were generated and used to identify the total number of genes expressed in each porcine sample and the DEGs among each comparison The DEGs between two samples were analysed based on an algorithm as previously described The P-value corresponds to a differential gene expression test in which False Discovery Rate FDR was used to determine the threshold of the P-value in multiple tests.
The Cluster 3. The R heatmap package 54 was applied to the analysis of Pearson and Spearman clustering. The functional classification of genes was performed using KEGG 55 pathway analysis. According to the location information of the reference genome Sscrofa All reactions were performed in triplicate. The relative expression values were then compared with the RNA-seq data. Tissues were added to cell lysis buffer Radio immunoprecipitation assay RIPA :phenylmethanesulfonyl fluoride PMSF in a ratio after they were ground in liquid nitrogen.
After washing, the membranes were incubated with a horseradish peroxidese-conjugated anti-rabbit antibody Santa Cruz; for 1. Testis sections from , and day-old Duroc and Meishan boars were deparaffinized and rehydrated with xylene and a graded ethanol series, respectively. Differentiation was assessed with 0. Then the sections were dehydrated using an ethyl alcohol series, cleared in xylene, embedded in paraffin wax and photographed using a microscope Olympus BX All experiments were repeated at least three times.
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