Morphophysiology of the epididymis and spermatozoa of the Wistar rat after sexual satiety

1. Introduction

Before referring to sexual satiety it is convenient to recall some aspects of the male rat sexual behavior. The males present, in a stereotyped way, three motor patterns during copulation, identified as: mounting, intromission and ejaculation. During the mounting pattern, the male with his forelimbs palpates the flanks of the female and performs pelvic movements back and forth on the female rump. The intromission pattern begins as the mounting pattern, it differentiates in that the last of the pelvic movements is deep and associated with the insertion of the erect penis in the vagina. Immediately the abrupt dismantling backwards follows (Pollak and Sachs, 1976). It is very common to observe a self-grooming of the genitals after the intromissions. After several mounts and interspersed interferences, the pattern of ejaculation occurs, characterized by the pelvic movement, which is deeper and more sustained than the intromission pattern, and it is associated with the deposition of semen in the vagina. Subsequently, the male raises the upper portion of the body by laterally extending the forelimbs. Finally, it slowly dismounts and self-grooms the genital region (Larsson, 1956).

2. The specificities of ejaculation

Ejaculation is defined as a male sexual physiological response (Eguibar et al., 2013). It is the vigorous exit of the seminal fluid by the urinary meatus (Lucio et al., 2014). It is conformed of two phases: the seminal emission and the seminal expulsion. The emission consists of a series of steps. Firstly, the confluence of the secretions coming from the accessory sexual glands (prostate, seminal vesicles and coagulating glands) in the prostatic urethra; secondly, the transport of the spermatozoa contained in the epididymal cauda, to the prostatic urethra by means of the contractions of the vas deferens (organs with layers of smooth muscle with circular and longitudinal disposition to intensify the contraction and thereby promote the transport of sperm) and thirdly, the closure of the urinary bladder neck and the external sphincter of the urethra to avoid retrograde ejaculation (Eguibar et al., 2013; Lucio et al., 2014).

The autonomic nervous system regulates the emission; the somatic, the expulsion (Eguibar et al., 2013). The seminal expulsion includes the sudden exit of seminal fluid (spermatozoa and glandular secretions) through the urinary meatus, produced by rhythmic contractions of the smooth muscle of the urethra and the striated muscles of the perineum including the ischiocavernosus and the bulbospongiosus muscles (Eguibar et al., 2013; Lucio et al., 2014). After the first ejaculation, the male presents a post-ejaculatory interval of approximately 10 minutes, in which the male remains without sexual activity (Eguibar et al., 2013). In the rat, most of the seminal plasma of the semen hardens forming the seminal plug (Blandau, 1945), which, by strongly adhering to the vaginal walls and the cervix, favor transcervical sperm transport. This transport occurs during the first 5 minutes after ejaculation (Matthews and Adler, 1977).

The post-ejaculatory interval prevents the male from withdrawing his own seminal plug before the transport of the spermatozoa previously deposited. The removal of the plug by another male in less than 5 minutes after ejaculation interrupts the sperm transport resulting in the minimum or null number of spermatozoa transported to the uterus (Matthews and Adler, 1977; Lucio et al., 2014). When the penis is inserted in the vagina, the spines of the penis hook in the seminal plug. So, when the penis is removed, the plug detaches from the vaginal walls and the cervix and approaches the vaginal orifice. To remove the seminal plug from the vagina, 3 to 4 interferences are enough.

3. Male sexual satiety

In the beginning of its study, sexual satiety was analyzed from a behavioral point of view (Larsson, 1956; Beach and Jordan, 1956); afterwards, from the neuroendocrinological and pharmacological perspective (Rodríguez-Manzo and Fernández-Guasti, 1994; Fernández-Guasti and Rodríguez-Manzo, 2003) and recently, it has been studied with an ecophysiological approach, that is, considering the influence of the environment on behavior and reproductive physiology. In sum, it studies not only the responses of an organism in its environment but also its physiology (Tlachi-López et al., 2012; Lucio et al., 2014).

The males of many species have sexual satiety not due to the physical fatigue of the individual, since their motor activity is similar to that presented by the nonsexually-satiated subjects; satiety is due to the loss of the sexual motivation for the female (Larsson, 1956; Phillips-Farfán and Fernández-Guasti, 2009). Sexual satiety can be achieved by repeated copulation with a same or a different female. Some males present a minimum of 5 ejaculations (Fernández-Guasti and Rodríguez-Manzo, 2003) and a maximum of 18 (Tlachi-López et al., 2012). The changes observed as the ejaculatory series progress are: a decrease in the number of intromissions, an increase in the latency of ejaculation and an increase in the duration of the post-ejaculatory interval (Larsson, 1956; Beach and Jordan, 1956; Rodríguez-Manzo and Fernández-Guasti, 1994).

4. The Coolidge effect

As mentioned before, male sexual satiety is due to the loss of sexual motivation to copulate with the same female. Hence, replacing it motivates the male again. Such a replacement represents a novel sexual stimulus so effective that it restores the copulatory behavior that includes ejaculation. The copulatory behavioral restoration is complete since it presents the mounting motor patterns, the intromission and the ejaculation patterns (Larsson, 1956; Beach and Jordan, 1956; Rodríguez-Manzo and Fernández-Guasti, 1994; Phillips and Fernández, 2009). The copulatory resumption with different females is known as the Coolidge effect (Wilson et al., 1963).

The term “Coolidge effect” was employed for the first time by Wilson et al. (1963) and its origin is due to an anecdote of the former President of the United States, Calvin Coolidge. It is said that one day, the President and his wife visited a poultry farm. Mrs. Coolidge was struck by the fact that one of the roosters copulated repeatedly, so she asked the manager of the farm how many times a day that male copulated. The answer was, “Dozens of times.” She asked him to communicate this fact to the President. When Calvin Coolidge was informed about the behavior of the rooster, he asked: “Does the male copulate every time with the same hen?” The answer was, “No, the cock copulates with different chickens each time.” The President nodded and said, “Please tell that to Mrs. Coolidge.” (Bermant, 1976).

5. Seminal expulsion in copula after sexual satiety

As it was previously mentioned, males sexually satiated during the Coolidge effect resume copulation reaching ejaculation with a different female. Does that mean that they expel semen? Our group has proved that sexually satiated male rats with Coolidge effect do not expel semen during the ejaculatory pattern, as it was verified revising the female reproductive ducts. In effect, Tlachi López et al. (2012) and Lucio et al. (2014) demonstrate and graphically describe that there is no seminal fluid in the uterine horns nor a seminal plug is present in the vagina. This shows that the motor pattern of ejaculation is independent of the genital response of seminal expulsion. It is worth mentioning that immediately after sexual satiety, 88% of males present the Coolidge effect, resuming copulatory activity.

Sexually satiated males maintain 44% of spermatozoa in the epididymal cauda compared to the amount found in non-sexually satiated male rats (Tlachi-López et al., 2012). Thus, satiated males have spermatozoa to continue expelling, but perhaps they do not because the accessory sex glands secretions are absent. It is mentioned that the ejaculate is composed of 1% of spermatozoa and 99% of seminal plasma. In addition, it has been shown that in the successive ejaculations the weight and size of the seminal plug diminish until it disappears, which means that the secretions of the seminal vesicles and the coagulant glands diminish to insignificance (Pessah and Kochva, 1975, Tlachi-López et al., 2012). If there are no glandular secretions, there is no seminal plasma, which prevents the transport of spermatozoa through the urethra until it exits through the urethral meatus. On the other hand, it is known that the spermatozoa in the epididymal cauda are those that are ejaculated. Its transport to the prostatic urethra during seminal emission is influenced by neuroendocrine mechanisms and perhaps also by the pressure exerted due to the high concentration of spermatozoa contained therein. This allows the confluence of the epididymal spermatozoa with the seminal plasma to form the semen that will be expelled during the ejaculation.

 6. Restoration of copulatory and ejaculatory behavior after sexual satiety

It is said that copulatory behavior is reestablished in sexually satiated male rats when the values of copulatory parameters are similar to those of non-sexually satiated males (Larsson, 1956). Copulatory reestablishment is also indicated when the males display at least 5 consecutive ejaculatory series, which happens up to 15 days after sexual satiety. They even manage to display 8 series, at 18-21 days after satiation (Beach and Jordan, 1956).

Most male rats that resume copulation immediately after reaching sexual satiety perform 1 to 3 ejaculatory series (Hsiao, 1969). However, only 33% of male rats execute a single ejaculatory series 24 hours after satiety; 63% after 3 days, and 100% after 7 days (Fernández-Guasti and Rodríguez-Manzo, 2003). It is important to note that all these percentages are based exclusively on the execution of the ejaculation motor pattern without the verification of the presence of ejaculate in the female ducts.

According to Dewsbury (1982), males are behaviorally potent when they physiologically are as well. However, there is evidence that males performing from 6 to 8 ejaculatory series, ejaculate very few spermatozoa and produce very small seminal plugs (Austin and Dewsbury, 1986).

With regard to ejaculation, it is common to consider that the seminal emission must precede the seminal expulsion, that is, that the stimulation of the prostatic urethra produced by the presence of sexual glandular secretions induces the second phase of ejaculation. However, the administration of guanethidine monosulfate that prevents seminal emission does not affect the execution of the ejaculatory pattern nor does it alter the activity of the bulbospongiosus muscles, whose contraction is required during seminal expulsion (Holmes and Sachs, 1991). This finding indicates that urethral stimulation is not necessary for the generation of rhythmic motor patterns associated with the ejaculatory response during copula (Holmes and Sachs, 1991). Sexually satiated male rats can be considered as a natural model to the evidence that the appearance of seminal emission is not critical to execute the ejaculation motor pattern.

The physiological cost of repeated copula affects the ejaculate and involves a drastic reduction on the weight and size of the seminal plug together with the absence of spermatozoa. The repercussion of copulating to sexual satiety abolishes expulsion (Tlachi-López et al., 2012; Lucio et al., 2014), which is due to the lack of accessory sex glands secretions (Pessah and Kochva, 1975; Purvis et al., 1986) added to the drastic decrease in the concentration of spermatozoa in the epididymis (Austin and Dewsbury, 1986; Toner and Adler, 1986; Tlachi-López et al., 2012). Therefore, semen will be gradually restored. In fact, our group found that after 5 days of the sexual satiety, males perform the ejaculation motor pattern without being able to expel semen (Lucio et al., 2014). Those same males semen tested 10 days after sexual satiety consists of a seminal plug of similar physical characteristics (weight and size) to that of males non-sexually satiated, although the plug does not adhere to the vaginal walls or the cervix. This lack of adhesion prevents transcervical sperm transport, causing the spermatozoa to remain inside the vagina (Lucio et al., 2014). The adhesion of the seminal plug depends on the secretions of the prostate (Tlachi-López et al., 2011). This suggests that the recovery of the prostatic secretion is slower than that of the seminal and coagulants involved in the formation of the plug.

The full recovery of accessory gland secretions appears 15 days after satiation, when the semen plugs are strongly attached to the vagina and facilitate sperm transport. However, its concentration represents a quarter of the normal spermatozoa concentration, i.e. around 6 million spermatozoa instead of 20-25 million (Lucio et al., 2014). As mentioned, these males performed an ejaculatory series at 5, 10 and 15 days after satiation. Interestingly, in the males who ejaculated once until 15 days after satiation, the spermatozoa concentration was 15 million (Lucio et al., 2014). This indicates that the seminal plasma provided by the different accessory sex glands is recovered before the spermatozoa concentration in which the testicle and the epididymis are involved (Lucio et al., 2013).

7. Epididymis and spermatozoa

The epididymis is a microtubular compact organ (according to the anatomical criterion), hence it is also known as an epididymal canal and is divided into four or more segments, depending on the species (Setchell et al., 2006; Garía-Lorenzana et al., 2007; Cornwall, 2009). In general terms, it is accepted, for most mammals, the existence of four anatomical regions: the initial segment as well as the caput, the corpus and the cauda (Setchell et al., 2006). In this work we focus on the caput, corpus and cauda, for being the conspicuous regions and being clearly defined anatomically. The caput is lined with pseudostratified cylindrical epithelium with microvilli while the corpus and cauda present a simple columnar epithelium with microvilli.

In the epithelium of the epididymis, there are different cell types: the main, the basal, the apical, the narrow, the clear and the halo (Figure 1). Some cell types are found in all anatomical regions of the epididymis (for example, the major cells) while others are characteristic of a region (for example, narrow cells are found mainly in the initial segment) (Setchell et al., 2006; Payal et al., 2011).

Figure 1. Photomicrographs of cross sections depicting: A) caput, B) corpus, and C) cauda of the rat epididymis. Different cell types can be seen, in which basophils and acidophils are noted as well. Each letter represents a cell type: P: major, B: basal, A: apical, C: clear, and H: halo (H-E, bars and original magnification: 500 μm and x400).

The main cells are responsible for absorbing fluids from the testicle and secreting proteins, serotonin, prostaglandins, amino acids, carbohydrates, lipids and electrolytes; in addition to intervening in the conversion of testosterone to dihydrotestosterone, a reaction catalyzed by the enzyme 5-alpha-reductase (Sullivan et al., 2007). The basal cells control the secretion of electrolytes from the main cells by the paracrine release of prostaglandin-E2. The apical and the narrow cells participate in the acidification of the seminal fluid; while the clear cells are endocytic and may be responsible for the clearance of lumen proteins. The immunological functions are related to the halo cells, since it has been demonstrated by immunocytochemistry that these cells are T lymphocytes or macrophages, according to the conditions of reproductive activity. The morphofunctional diversity of cell types of the epithelium determine the necessary conditions for the spermatozoa to reach maturity, acquire motility, as well as the ability to recognize, fuse and fertilize the oocyte (Setchell et al., 2006; Sullivan et al., 2007).

It is important to emphasize that the distribution, proportion and functions of the described cell types differ between the different segments of the epididymis. This situation generates microenvironmental domains that provide spermatozoa with different conditions (nutrients, ions and chemical compounds) that maintain sperm survival as they travel through the organ (Setchell et al., 2006). Different types of binding are present in the epididymal epithelium: occluders, adherents and communicants that allow the formation of a hemato-epididymal barrier which, in addition to protecting spermatozoa from the adverse effects associated with the intake or use of drugs and other harmful compounds that may come from the diet, prevents direct contact of the immune cells with the spermatozoa, thus preventing their autoimmune destruction (Setchell et al., 2006; Cornwall, 2009; Payal et al., 2011).

In addition to epithelial tissue, the wall of the epididymis has a middle layer consisting of myoid cells and an outer layer called adventitia, formed by loose connective tissue (Setchell et al., 2006). In the mature organ, the fundamental interstitial substance of the connective tissue (with its molecular components: water ions and proteoglycan), participates as a barrier that limits the diffusion of molecules, from the blood vessels to the epithelial tissue (Sullivan et al., 2007).

On the other hand, the interstitial space has a cellular component constituted by cells of the nervous, neuroendocrine, vascular and fibroblastic lineages that provide innervation, nutritional supply (through the blood vessels) and participate actively in the control of local secretion processes, absorption and remodeling of the fibrillar components and the extracellular matrix. Especially, the mast cells, which stem from the bone marrow, are particularly abundant in the epididymis and are apparently involved in the processes of tissue remodeling that occurs during the involution-recrudescence cycle, which occurs in the testicle and the epididymis of mammals with seasonal reproduction (Setchell et al., 2006; Cornwall, 2009; Amann et al., 1993).

It is important to note that the time for the spermatozoa to travel through the cephalo-caudal extension of the epididymis varies between species. In the rat it takes from 8 to 12 days depending on the strain. The main propulsion force of the spermatozoa within the lumen of the epididymis is of neuromuscular origin, subject to the spontaneous rhythmic contractions generated in the wall in response to androgens, the intrinsic modulation of the neurons, the hypogastric nerve activity and the action of various neurotransmitters on the myoid cells of the epididymis (Sullivan et al., 2007; Setchell et al., 2006; Robaire et al., 2006). Therefore, the spermatozoa that are ejaculated are found particularly in the epididymis cauda (Setchell et al., 2006).

When spermatozoa leave the testicle, they are not yet able to fertilize the oocyte. They acquire this capacity during their transit through the epididymis, a process in which androgens are required, which are responsible for maintaining the structure and function of the epididymis (Robaire and Hamzeh, 2011; Seenundun and Robaire, 2007). Immediately after sexual satiety, serum testosterone increases significantly, however, it is reestablished at baseline 24 hours after sexual satiety (3.97 ± 1.03 ng/ ml) (Bonilla-Jaime et al., 2006), and remains the same at 48 hours (2.03 ± 0.28 ng/ml), 72 hours (3.80 ± 0.86 ng/ml) and 7 days after sexual satiety (2.28 ± 0.37 ng/ml) (Romano-Torres et al., 2006). It is unknown whether the epididymis has morphological changes, as well as local testosterone levels, after sexual satiety (Robaire et al., 2007; Hamzeh and Robaire, 2009).

8. Epididymal sperm maturation

Sperm maturation occurs in the epididymis. This process is defined as the morphological, physiological and biochemical changes that occur in spermatozoa, conferring the capacity to carry out the acrosome reaction and fertilize the oocyte. Sperm maturation develops when spermatozoa migrate from the caput to the cauda of the epididymis (Cooper and Ching, 2006). Among these modifications is the progressive motility that spermatozoa acquire by passing through this organ (Robaire et al., 2006), e.g. in rodents, sperm motility is obtained by the highest percentage of spermatozoa in the caput, more than in other regions of the organ (Avilés, 2011).

All these modifications are necessary to acquire the essential potential for fertilization processes, e.g. training and the acrosome reaction in the female reproductive tract. As mentioned, the processes involved in sperm maturation include biochemical events that require the presence of components secreted by different epithelial cell types, e.g. they secrete proteins that allow sperm maturation (Rodríguez-Manzo et al., 2011). Two essential processes are changes in the plasma membrane: glycosylation (Jiménez and Merchant, 2003) and phosphorylation (Lewis and Aitken, 2001, Rodríguez-Tobón et al., 2015).

Protein glycosylation is the addition of carbohydrates to a protein that will be part of the cell surface. It works as an indicator to determine its intracellular distribution and its final place of functioning, including the pattern of secretion in the event that its function is exercised outside of the cell (Rosado, 2000). In the spermatozoon, the glycoproteins, carnitines and glycerophosphocholine will be integrated into the plasma membrane (Avilés, 2011; Rodríguez-Manzo et al., 2011), permitting sperm-ovule interaction.

While phosphorylation is the addition of a phosphate group, it is the basic mechanism of energy transport, as well as of the regulation of proteins and enzymes through post-translational modifications (Laserna, 2009). Phosphorylation of proteins is important in extracellular signal transduction, intracellular transport and cell cycle progression. It is regulated by the activity of proteins-kinases and phosphatases, allowing cells to activate or disable the function of other proteins. This phosphorylation occurs in the serine, threonine and tyrosine residues of the proteins, with tyrosine phosphorylation being the most important. In spermatozoa it is associated with flagellar proteins, since they play an important role in motility changes (Aviles, 2011, Laserna, 2009). These intracellular processes involved in sperm maturation have not been studied in males with sexual satiety or in the period after satiety that includes up to the morphofunctional recovery of the epididymis and consequently, the fertilizing capacity of the spermatozoa.

9. The morphophysiology of the epididymis in sexual satiety

It was previously mentioned that after sexual satiety, serum testosterone is not modified (Romano-Torres et al., 2006). Thus the question arises of what happens with the morphophysiology of the epididymis after sexual satiety. Our work group analyzed the different parameters of the epididymal morphology (cytology of the cells, type of epithelium, cell count and morphometry) and of the sperm parameters (viability, concentration and morphology) of males of the control group, immediately to the sexual satiety and at 4, 8, 12, 16, 20, 24 and 28 days after sexual satiety. It was found that the weight of the epididymis in its different regions, as well as the structural, biochemical and functional cytological characteristics of the cell types that make up the epididymal epithelium present variations (in the affinity to the dyes used in the histological analysis, in the form of cells, cell types, height of the epithelium) that denote changes in the activity of synthesis and secretion of cellular products.

Among the most important cellular changes are those involved in sperm maturation, which explains that changes in concentration, viability and sperm morphology are observed in the period after sexual satiety. These changes can be caused by alterations in the intraepididymal androgen concentration as well as by the activity of enzymes, such as aromatase, and of the androgen receptors in the cells of the epididymal epithelium and even to the innervation of the tissue components of the epididymal ducts wall.

Glossary

Accessory sex glands: Organs anatomically associated with the male reproductive system that synthesize the secretions that form the seminal plasma.

Amino acids: Molecules that contain an amino group at one end of the molecule and a carboxylic acid group at the other end. They are the units that form proteins.

Bulbospongiosus muscle: Its fibers originate in the penile bulb (erectile tissue of the base of the penis) and are inserted into the cavernous body of the penis (erectile tissue of the body of the penis). It is a paired muscle.

Carbohydrates: Most abundant biomolecules composed of carbon, hydrogen and oxygen. They function as an energy source,and as structural units. They are precursors of the synthesis of other biomolecules.

Myoid cells: Morphological and functional units that surround the seminiferous tubules causing rhythmic peristaltic contractions that facilitate the passage of sperm and testicular fluids through the seminiferous tubules.

Copula: Set of motor patterns accompanied by genital responses that culminate with ejaculation.

Bladder neck: Set of muscles that join the urinary bladder with the urethra. They contract to maintain urinary continence and relax during the micturition reflex.

Dihydrotestosterone: Biologically active metabolite of testosterone, also called androgenic hormone; it is synthesized by the testicles and the prostate, mainly. Its important contribution is in the development of male sexual characteristics.

Glycosylation: The process of adding carbohydrates to a protein that will be part of the cell membrane. In the spermatozoon, the glycoproteins, carnitine and glycerophosphocholine are integrated into the membrane.

Guanethidine monosulfate: Adrenergic drug that prevents the seminal emission phase.

Hypogastric: Part of the abdomen located below the umbilical region and between the iliac fossae.

Microtubules: Internal components of the cytoskeleton of eukaryotic cells formed by alpha and beta- tubulin dimers. They participate in the function of ciliated and flagellated cells.

Motor pattern: Easily identifiable stereotyped movement that can be recorded and quantified.

Paracrine: Chemical communication between cells of immediate proximity and different cell lines.

Perineum: Anatomical region where the exit orifices of the urogenital and digestive system are located. Also known as pelvic floor.

Phosphorylation: Synthesis of ATP (adenosine triphosphate) from ADP (adenosine diphosphate) and phosphate to obtain energy.

Prostaglandins: Molecules of lipid character derived from fatty acids of 20 carbons. They constitute a family of cellular mediators, with diverse effects, often opposed.

Prostate: Accessory sexual gland located at the base of the urinary bladder surrounding the proximal portion of the urethra. It provides zinc and fructose necessary for sperm motility.

Protein kinase: Transducer enzyme of cellular and/or tissue signals that modifies other molecules (proteins) through phosphorylation.

Serotonin: Neurotransmitter that is synthesized mainly in the brainstem raphe nuclei projected to different areas of the brain and spinal cord. It regulates some sensory, motor and behavioral functions in mammals.

Testosterone: Androgen synthesized from cholesterol by testicular Leydig cells. It is also synthesized in the adrenal cortex and in the ovary. It has metabolic sexual actions on the pituitary, stimulants on erythropoiesis and antineoplastic.

Transcervical: That crosses the cervix of the vagina to the uterus.

Prostatic urethra: Portion of the urethra surrounded by the prostate gland.

Urethral meatus: Orifice at the free end of the penis where urine and semen are expelled.

Urethral mucosa: A layer of epithelial and conjunctive tissue that lines the inner walls of the urethra.