Introduction

The union of male and female gametes creates offspring. The production of these vital reproductive cells occurs in the testis and the ovary during spermatogenesis and oogenesis, respectively.[1] The primary male reproductive organs, the testes, are located in the scrotum and produce sperm cells and the primary male hormone, testosterone. As mentioned above, spermatogenesis is the process by which sperm cells are produced; germ cells give rise to haploid spermatozoa. Sperm production takes place inside the seminiferous tubules, which are a convoluted cluster of tubes located inside the testes. Testosterone is produced by Leydig cells, which surround the seminiferous tubules. 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 3 steps. The first step involves mitotic cell division, which 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 the production of mature, 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 3 steps represent the foundation of spermatogenesis. Functional abnormalities may occur in any of them, leading to the entire process failing. 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, studying 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 need to produce an infinite number of gametes increases the specific requirements for spermatogenesis. The reproductive lifecycle of the male requires a large amount of stem cell production. A wide array of specific progenitor cells is necessary to produce enough gametes to ensure fertilization. Certain genes specific to spermiogenesis are required for sperm differentiation and for the acquisition of sperm mobility. The continuous pool of spermatozoa becomes readily available through advanced 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 through self-renewal and to form the progenitor cells required for spermatogenesis. Germ cells require constant nutrition to differentiate into mature sperm capable of fertilization. The absence of these factors can ultimately cause spermatogenesis to fail, leading to a lack of sperm production.[4][5]

Spermatogenesis is characterized by a high rate of germ cell attrition, with a substantial proportion of developing germ cells undergoing apoptosis before reaching full maturation. The loss of germ cells occurs quite often. Also, the ejaculate can have extremely high numbers of malformed spermatozoa. Apoptosis or degeneration results in the loss of about 75% of the germ cells that have developed. 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 few decades, and specific factors affecting embryonal development seem to be the cause. These factors include prenatal influences such as hormones, drugs, radiation, metabolites in drinking water, and the mother's nutrition. Moreover, the spermatogenic process of the testis is affected by increased temperatures. These negative influences lead to reduced spermatogenesis, manifested as fewer mature spermatids or malformed spermatids.

Additionally, these influences may disrupt the meiotic process. Spermatogenesis may arrest after the formation of primary spermatocytes, and apoptosis of spermatogonia may occur. Rescue of spermatogenesis may arise if the spermatogonia survive. If they do not survive, spermatogenesis comes to a halt, and seminiferous tubules appear as shadows.[3] Testicular biopsies evaluate disturbances in spermatogenesis through histological analysis. A suitable technique is semithin sectioning of material embedded in epoxy resin. The excellent preservation of the cells greatly facilitates the evaluation of their specific details.[3]

Structure

The human testes, which are ellipsoid-shaped, are 2 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 the thin septa 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. The rete testis is a network of tubules found in the hilum of the testicle and functions 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 show increasing numbers of lipid droplets over time. Therefore, the increasing numbers of lipids correlate with the advancing age of the individual.[3] 

Other functions attributable to Sertoli cells include providing nutrition for germ cells, delivering mature spermatids into 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), and interacting with the intertubular 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 with 5 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 is usually about 8 micrometers in thickness. Alteration of spermatogenesis can increase the width to 12 micrometers due to connective tissue.[3]

Leydig cells, which are most prominent in the inter-tubular space, form clusters surrounding the capillaries. They produce and secrete 1 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 demonstrate that testosterone production occurs only 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 3 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 4 daughter haploid cells, also known as spermatids. For every diploid spermatocyte, meiotic divisions produce 4 haploid spermatids.[1][5]

Mitotic cell division: diploid spermatogenic stem cells differentiate into 2 sets of diploid primary spermatocytes, also known as tetraploid cells. The largest germ cells in the germinal epithelium are the primary spermatocytes, which have the largest cell nuclei.[3]

Meiotic Cell Division I

The division begins with the leptotene stage of prophase. The stage occurs in the germinal epithelium, specifically the basal compartment. Spermatocytes enter the adluminal compartment after reaching the Sertoli cell barrier. Here, prophase continues through the zygotene, pachytene, and diplotene stages. These stages include DNA reduplication, chromosome condensation, and homologous chromosome pairing, with crossing-over occurring. Each set of diploid primary spermatocytes differentiates into 2 haploid secondary spermatocytes, where the total number of chromosomes is reduced to half.[3][6]

Meiotic Cell Division II

Each haploid secondary spermatocyte gives rise to 2 haploid spermatids, yielding 4 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 are called spermatozoa at this point. Spermatozoa have a unique shape, which is essential in their movement to the female gamete. The condensed nucleus and the presence of an acrosome, which are needed to establish contact with the female gamete, provide them with their unique shape. They exhibit extensive motility because they possess a single 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 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 regulate myofibroblast contractility to ensure proper sperm transport. Intrinsic factors also play a vital role in regulating blood flow 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 releasing 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 and 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 2 primary functions in the human body: the production of male gametes (sperm cells) and testosterone, 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 10, the bipotential gonad is first visualized as a thickening of the mesonephros located on the ventral side. Gender specificity acquisition occurs over the next 2 days, primarily driven by “SRY” gene expression. Expression of this gene allows cells to differentiate into Sertoli cells, which are testicular cells. In contrast to females, in the absence of SRY gene expression, granulosa cells form in a developing ovary. For sex determination to be complete, male testis formation requires primordial germ cells to interact with embryonic Sertoli cells, myoid cells, and Leydig cells. This process occurs by embryonic day 12.5. Primordial germ cells appear within the epiblast at embryonic day 6, where they undergo cell migration. They arrive and infiltrate the developing gonadal ridge at embryonic day 11. Once cells are in close proximity, they can form seminiferous cords, which eventually coalesce into 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, this population of cells enters 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, they eventually migrate to the periphery after birth. Several essential processes occur at this location, leading to morphologically distinct spermatogonia.

Oocytes in females and sperm in males are derivatives of primordial germ cells. Their production depends on retinoic acid, a derivative of vitamin A. 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 retinoic acid signaling. 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 are 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 rather than healthy spermatogonia. They can be seen on histological sections, differing from spermatogonia due to their noticeably larger size, prominent nucleolus, increased glycogen content, and characteristic peripheral border. The presence of these neoplastic cells contributes to carcinoma in situ and can lead to hypospermatogenesis. Carcinomatous cells characterize the stem cell population in many, if not most, germ cell tumors. Examples include seminomatous and 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, leading to detachment of the remaining spermatogonia, which then enter the tubular lumen. As cancer cells proliferate, they are eventually released into the tubular lumen or penetrate the lamina propria of the seminiferous tubules, forming intertubular 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 carcinoma-in-situ cells. A score-count system evaluates the histology of cancer cells and other techniques used to assess protein and mRNA expression. Testicular biopsies are also recommended and should be completed 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 halt at the primary spermatocyte stage, preventing morphological changes in the cells. Primary spermatocytes are seen to border the lumen of seminiferous tubules. They do not develop further, leading to cell disintegration and a failure to produce spermatids.[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 effects of mutations in these genes on spermatogenesis remain under investigation.[9]

A study using testicular histology to determine the effects of aging on spermatogenesis revealed various alterations, including basal membrane thickening, decreased germinal and Sertoli cell numbers, and individual variation. 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 2 haploid gametes. Currently, there are many different assisted reproduction methods available, developed based on our knowledge of spermatogenesis. Identification of mature spermatids and spermatozoa is based on specific morphological features.[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.

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Disclosure: Samah Suede declares no relevant financial relationships with ineligible companies.

Disclosure: Ahmad Malik declares no relevant financial relationships with ineligible companies.

Disclosure: Amit Sapra declares no relevant financial relationships with ineligible companies.