Introduction

The union of male and female gametes creates offspring. The production of these vital reproductive cells occurs in the testis and ovary during the processes of spermatogenesis and oogenesis, respectively.[1] The primary male reproductive organs, the testes, are located inside the scrotum and function to produce sperm cells as well as the primary male hormone, testosterone. As mentioned above, spermatogenesis is the process by which sperm cell production occurs; the germ cells give rise to the haploid spermatozoa. Sperm production takes place inside the seminiferous tubules, which is a convoluted cluster of tubes located inside the testes. Testosterone production occurs in cells surrounding the seminiferous tubules, called Leydig cells. After being formed, sperm cells travel outside of the tubules into the epididymis, where they mature and prepare for ejaculation.

The complex process of spermatogenesis occurs in three steps. The first step involves mitotic cell division that allows the early cell stage, spermatogonia, to multiply. The second step requires meiosis, in which the diploid cells form haploid cells. A division occurs until a round spermatid formation occurs. The final stage of spermatogenesis includes spermatozoa production, mature and motile sperm cells, from round spermatids, through a process called spermiogenesis.[2] 

Diminished fertility or infertility may result from a decrease in spermatozoa number, alteration in shape, and inefficient motility.[3]

The three steps represent the foundation of spermatogenesis. Functional abnormalities may occur in any one of them, which can cause the entire process to fail. These abnormalities can lead to defective or reduced sperm production. In more severe conditions, a complete absence of spermatozoa can result, leading to infertility. Therefore, we must expand our knowledge of spermatogenesis as a whole to provide essential information regarding the regulatory mechanisms. The testicular environment is complex; therefore, the study of spermatogenesis can be quite tricky in most species. To achieve this understanding, experimental studies completed in rodents and primates are the cornerstone of this crucial knowledge.[2]

Issues of Concern

The necessity for the production of an infinite number of gametes increases the specific requirements needed for spermatogenesis to occur: The reproductive lifecycle of the male requires a large amount of stem cell production. A wide array of particular progenitor cells is necessary for the production of enough gametes to establish fertilization. Certain genes specific to spermiogenesis are required for the differentiation of sperm as well as acquiring sperm mobility. The continuous pool of spermatozoa becomes readily available by an advanced level of control and organization. One of the principal functions in male reproduction is carried out by the spermatogonia stem cells. Spermatogonia need stem cells to maintain their numbers by self-renewal and form the necessary progenitor cells required to proceed with spermatogenesis. Germ cells need constant nutrition to be able to differentiate into mature sperm that are capable of fertilization. Unavailability of these factors can cause spermatogenesis to fail ultimately and lead to sperm cell production.[4][5]

Spermatogenesis is far less efficient in terms of quality management. The loss of germ cells occurs quite often. Also, the ejaculate can have extremely high numbers of malformed spermatozoa. Apoptosis or degeneration allows for the loss of about 75% of the developed germ cells. Only 25% of the germ cells reach the ejaculate, and research reveals that about 50% are malformed. Therefore, the spermatogenic potential that is accessible for reproduction is about 12%.[3]

Recent reports have noticed a decline in the spermatozoa concentrations in the ejaculates of healthy males. This decline has occurred over the last decades, and specific factors affecting embryonal development seem to be the cause. These factors include prenatal influences such as hormones, drugs, radiation, metabolites in the drinking water, and nourishment of the mother. Moreover, the spermatogenetic process of the testis is affected by increased temperatures. These negative influences lead to a reduction in spermatogenesis, which manifests as a reduction in the number of mature spermatids or the formation of malformed spermatids.

Additionally, these influences may cause the process of meiosis to be disturbed. An arrest of spermatogenesis may occur after the creation of primary spermatocytes, and apoptosis of spermatogonia can occur. Rescue of spermatogenesis may arise if the spermatogonia survive. If they do not survive, spermatogenesis comes to a halt, and seminiferous tubules will appear as shadows.[3] 

Testicular biopsies evaluate the disturbances of spermatogenesis through histological sections. A suitable technique is known to be semithin sectioning of material embedded in epoxy resin. The excellent preservation of the cells much helps the evaluation of the specific details of the cells.[3]

Structure

The human testes, exhibiting an ellipsoid shape, are two reproductive organs that reside in a sac called the scrotum. Each testis is about 2.5 x 4 cm in diameter, surrounded by an active capsule of connective tissue called tunica albuginea. The parenchyma of the testis is divided roughly into 370 conical lobules by thin septula testis. Within the lobules are the seminiferous tubules as well as inter-tubular tissue. This tissue contains groups of Leydig cells and further cellular elements.[3]

The seminiferous tubules consist of entwined loops whose ends unfold into the open space of the rete testis. Rete testis is a network of tubules found in the hilum of the testicle and function to carry collected sperm to the efferent ducts of the epididymis as the seminiferous tubules secrete it. The germinal epithelium constitutes the seminiferous tubule. The invagination of Sertoli cells houses many cells. These cells include germ cells in different developmental stages such as spermatogonia, primary and secondary spermatocytes, and spermatids. The tight junctions of cellular membranes connect Sertoli cells. The specialized zones of tight junctions form the blood-testis barrier. Germ cells pass the hurdle during maturation, where they get protection from the diffusion of external substances. Sertoli cells are known to be the “biological clock” of the testis. When visualized in histological sections, they manifest rising amounts of lipid droplets with time. Therefore, the increasing numbers of lipids correlate to the advancing age of the individual.[3] 

Other functions attributable to Sertoli cells include providing nutrition for the germ cells, delivering mature spermatids to the tubular lumen, regulating spermatogenesis by producing endocrine and paracrine substances, maintaining the epithelia of the efferent ductal system by secreting ABP (androgen binding protein) as well as interacting with the inter-tubular Leydig cells. The lamina propria of seminiferous tubules, also known as the peritubular tissue, constitutes the basement membrane of the basal lamina and functions to surround the cellular elements. It consists of connective tissue intermingled around five layers of myofibroblasts. These myofibroblasts play a vital role in the peristaltic contraction of seminiferous tubules to allow for the transport of immotile spermatozoa to the hilum of the testis, the rete testis. Peritubular tissue usually is about 8 micrometers in thickness. Alteration of spermatogenesis can increase the width to 12 micrometers by connective tissue.[3]

Leydig cells, which are most prominent in the inter-tubular space, form clusters surrounding the capillaries. They produce and secrete one of the essential male reproductive hormones, testosterone. Testosterone is vital for male reproduction as it activates the hypophyseal testicular axis. It is crucial for the development of male genital organs as well as masculinization and formation of the secondary sex characteristics. Testosterone plays a significant role in initiating, processing, and maintaining spermatogenesis. Hormone production does not correlate with the number of Leydig cells. Immunohistochemical investigations prove that the creation of testosterone only takes place in a few Leydig cells despite their increased number. An increased number of cells occurs in hyperplasia or tumors.[3]

Function

Spermatogenesis involves the following three complex integrated processes initiated at the onset of puberty:

Meiosis

Male fertility requires the formation of millions of gametes. Production of functional gametes or sex cells requires a single cell to undergo cell division to reduce the number of chromosomes by half. This process is known as meiosis. Meiosis occurs twice, thereby creating four daughter haploid cells, also known as spermatids. For every diploid spermatocyte, meiotic divisions produce four haploid spermatids.[1][5]

Mitotic cell division: diploid spermatogenic stem cells differentiate into two sets of diploid primary spermatocytes, also known as tetraploid cells. The largest germ cells found in the germinal epithelium are the primary spermatocytes, containing the largest cell nuclei.[3]

Meiotic Cell Division I

The division begins with the leptotene stage of prophase. The stage takes place in the germinal epithelium, more specifically, the basal compartment. Spermatocytes enter the ad luminal compartment after reaching the Sertoli cell barrier. Here, further prophase stages continue, and these include the zygotene, pachytene, and diplotene stages. These stages see DNA reduplication, chromosomes condensation, and homologous chromosomes pairing, as crossing over will occur. Each set of diploid primary spermatocytes differentiates into two haploid secondary spermatocytes, where the total number of chromosomes gets reduced to half.[3][6]

Meiotic Cell Division II

Each haploid secondary spermatocyte differentiates into two haploid spermatids, therefore, resulting in four haploid cells. This process occurs quickly, and no DNA replication takes place.[3]

Spermiogenesis

This process is the final phase of spermatogenesis. Spermatids mature and form spermatozoa, fully differentiated sperm cells. This stage ends when the mature cells leave the germinal epithelium. These free cells, at this point, are called spermatozoa. Spermatozoa have a unique shape, which is essential in their movement to the female gamete. The condensed nucleus and the presence of an acrosome needed to establish contact with the female gamete provide them with their unique shape. They exhibit extensive motility as they contain a connected flagellum. As they transform into mature sperm, spermatids undergo a wide array of morphological changes.[3][5] It takes about 30 to 40 days for spermatogenic stem cells to generate spermatozoa.[4] Spermatogenesis is a process that occurs in the seminiferous tubules, and many internal and external factors regulate it. Intrinsic regulation requires the production and release of testosterone, neuroendocrine substances, and growth factors secreted by Leydig cells. They communicate with nearby Leydig cells, blood vessels, peritubular tissue of the seminiferous tubules, and Sertoli cells. They maintain the trophic factors of these cells and participate in the regulation of peristalsis in the seminiferous tubules. These growth factors and neuroendocrine substances influence the contractility of myofibroblasts to ensure the proper transport of spermatozoa. Intrinsic factors also play a vital role in regulating the flow of blood in the intertubular microvasculature. This process is quite intricate and is mainly investigational in laboratory animals. In humans, it is still unclear.[3]

Extrinsic regulation of spermatogenesis requires stimuli from the hypothalamus and hypophysis. The hypophysis awaits a signal from the hypothalamus to begin the release of LH (luteinizing hormone). This signal is the pulsatile secretion of the hormone GnRH (gonadotropin-releasing hormone). The release of LH stimulates the Leydig cells to produce testosterone. Testosterone has a significant effect on spermatogenesis as well as other functions throughout the body. Sertoli cells are stimulated by FSH (follicle-stimulating hormone), an important signal that allows for the maturation of germ cells. Sertoli cells secrete inhibin that is involved in the feedback mechanism.[3]

Histochemistry and Cytochemistry

The male reproductive organs, the testes, serve two primary functions in the human body, including the production of the male gametes, sperm cells, and testosterone, which is the primary male sex hormone. Located inside the testes are groups of convoluted tubules called seminiferous tubules, where sperm cell production takes place.

At embryonic day ten, the bipotential gonad is first visualized as a thickening of the mesonephros located on the ventral side. Gender specificity acquisition occurs over the next two days, primarily driven by “SRY” gene expression. Expression of this gene allows cells to differentiate and become the Sertoli cells, which are cells of the testis. In contrast with females, if SRY gene expression is absent, granulosa cells will form in a developing ovary. For sex determination to be complete and for male testis formation requires primordial germ cells to interact with embryonic Sertoli cells, myoid cells, and Leydig cells. This process occurs by embryonic day twelve and a half. Primordial germ cells appear within the epiblast at embryonic day six, where they undergo cell migration. They arrive and infiltrate the developing gonadal ridge at embryonic day 11. Once the cells are in close approximation to one another, they can form the seminiferous cords, which eventually form the seminiferous tubules.[4]

The primordial germ cells undergo mitotic proliferation within the cords of an embryonic testis. They are then called prospermatogonia or gonocytes. After replication of this population of cells, prospermatogonia enter a dormant non-proliferative phase until birth in the rodent. The initial location of the prospermatogonia is closer to the center of the seminiferous cords; however, eventual migration occurs to the periphery after birth. Several essential processes arise at this location, producing the morphologically distinct spermatogonia.

Oocytes in females and sperm in males are derivatives of primordial germ cells. Their production is dependent on a derivative of vitamin A, known as retinoic acid. During fetal development in females, meiosis is initiated by germ cells in the ovary in response to retinoic acid. However, in the testis, germ cells do not receive the signal from retinoic acid. Hence meiosis is not initiated until after birth. The period corresponds to the resumption of male germ cell proliferation and the transition to spermatogonia. Eventually, spermatogenesis occurs, and haploid spermatozoa get created.[7] Sertoli cells produce and secrete an essential regulatory factor called glial cell-derived neurotrophic factor (GDNF). GDNF is critical to the survival and proliferation of the undifferentiated spermatogonia.[4]

Pathophysiology

Occasionally, the seminiferous tubules may contain tumor cells in the basal compartment instead of healthy spermatogonia cells. They can be seen on histological sections differing from spermatogonia due to their noticeable larger size, prominent nucleolus, increased glycogen content, and characteristic peripheral border. The presence of these neoplastic cells attribute to carcinoma-in-situ and can lead to hypospermatogenesis. The carcinomatous cells characterize the stem cell population for many and most germ cell tumors. Examples include seminomatous as well as teratomatous tumor types. During active spermatogenesis, the tubules may give rise to sporadic tumor cells. However, spermatogenesis ceases as the cancer cells increase in number, and this results in detachment of the remaining spermatogonia, which then enters the tubular lumen. With the continuous proliferation of the cancer cells, they are also eventually released into the tubular lumen or penetrate the lamina propria of the seminiferous tubules leading to the formation of inter-tubular tumor cell clusters.[3] PLAP, which is placental-like alkaline phosphatase, is an immunohistochemical marker used to diagnose preinvasive carcinoma in situ. It is manifested exclusively in these carcinomas in situ cells. A score-count system evaluates the histology of the cancer cells and other techniques used for protein and mRNA expression. Testicular biopsies are also recommended and should undergo completion in specialist centers.[8]

Meiosis is a convoluted process that is vulnerable to many faults and defects. Apoptotic spermatocytes can arise in the process and are known to be frequent. Megalospermatocytes, which are very large spermatocytes, can sometimes appear. In these cells, homologous chromosomes fail to pair in a process called asynapsis, causing the cells to become abortive. Moreover, spermatogenesis can come to a halt at the stage of primary spermatocytes, stopping the morphological changes from occurring in the cells. Primary spermatocytes are seen to border the lumen of seminiferous tubules. They will not develop any further, which will lead to the disintegration of the cells and lack of spermatids production.[3]

The structure of the sperm tail closely resembles the motile cilium in that the axoneme has a 9+2 microtubular arrangement. Therefore, genetic defects detected in motile cilia can significantly affect the formation of the sperm tail. A genetic disease called PCD (primary ciliary dyskinesia) due to the malformation of motile cilia causes pulmonary disease, increased risk of infections, and male infertility. Many genes are associated with PCD; however, the exact effect these mutated genes have on spermatogenesis is still investigational.[9]

A study on using testicular histology determining the effects of aging on spermatogenesis showed various alterations, including basal membrane thickening, a decrease in the germinal and Sertoli cell number, as well as an exhibition of individual variations. Upon examination of post-meiotic cells, the aneuploidy rate was higher than average during the arrest of spermiogenesis. However, they concluded that spermatogenesis could still be possible until the male is 95 years old.[10]

Clinical Significance

In diploid organisms, sexual reproduction requires the union of two haploid gametes. Currently, there are many different methods available for assisted reproduction, founded as a result of the knowledge we have on spermatogenesis. The identification of mature spermatids and spermatozoa occurs through specific morphological processes.[3] Induction of pregnancies can occur through specialized techniques that extract spermatozoa from testicular tissue, followed by injection into the cytoplasm of the ovum. These techniques can significantly enhance and develop the assisted reproductive technologies available today.