Normal Testicular Function Research Articles

101. THE RELATIONSHIP BETWEEN SERTOLI CELL STATUS AND IDIOPATHIC MALE INFERTILITY

The cornerstone of normal adult testicular function is a mature Sertoli cell population. The maturational switch for Sertoli cells occurs at puberty, where immature Sertoli cells differentiate into a mature population that hold the necessary architectural and functional characteristics to regulate spermatogenesis (1). Data from rodent models (2, 3) suggest a relationship between Sertoli cell immaturity and infertility, however clinical data confirming this relationship is limited. We postulate that adult Sertoli cells in the infertile human testis display an immature status, with more severe disruptions of spermatogenesis associating with a greater extent of Sertoli cell immaturity. Using testicular biopsy samples obtained from fertile men (n = 3) and infertile patients (n = 6/group) displaying meiotic arrest (MA) and Sertoli cell only (SCO) syndrome, we sought to survey the status of Sertoli cell populations. All samples were immunofluorescently probed for three hallmark features of adult Sertoli cell maturation; organisation of the inter-Sertoli tight junctions, expression of the androgen receptor and proliferative ability. Differences between groups were quantified using stereology. The results show that the majority of infertile patients display highly disorganised tight junctions, a feature not seen in fertile men, however surprisingly no difference in the extent of tight junction disruption was observed between MA and SCO. Preliminary data also show that some component of the Sertoli cell population in MA and SCO patients was non-functional and proliferative. These results suggest that the Sertoli cell population in men suffering from idiopathic infertility present an abnormal maturational status that is independent of the extent of spermatogenic disruption. Moreover, this study supports the growing body of evidence proposing that the adult Sertoli cell population is not a homogenous, terminally differentiated population, and suggests that the failure of Sertoli cells to reach or maintain their mature status may be the cornerstone of abnormal adult testicular function. (1) Sharpe R.M. et al., 2003, Reproduction, 125: 769.(2) Tarulli G.A. et al., 2006, Biology of Reproduction, 74: 798–806.(3) Tarulli G.A. et al., 2008, Reproduction, 135: 867–877.

Testicular function and fertility in men with homozygous alpha-1 antitrypsin deficiency.

Fertility and testicular function were studied in eight men with severe homozygous (Pi ZZ variant genotype) alpha-1 antitrypsin (AAT) deficiency. Age- and marital duration standardized fertility, clinical androgenic features, mean testicular volume, plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and semen analysis were all normal apart from a reduction in semen volume. Mean plasma total and free testosterone were elevated and the percentage free testosterone reduced compared with age-matched, healthy fertile controls indicative of increased sex-hormone binding globulin (SHBG) levels representing an early marker for subclinical hepatic dysfunction associated with AAT-deficiency. In view of the preservation of normal fertility and testicular function despite chronic respiratory disease and premature death with deleterious AAT gene variants, it is proposed that the high prevalence of genetic polymorphism in the AAT protein may be maintained by the chronological asynchrony of the periods of maximal male reproductive activity (40 years) and the late onset (greater than 40 years) of symptoms in severe AAT deficiency rather than by any balance between reduced reproductive fitness of homozygotes and heterozygote advantage.

Effect of Childhood Acute Lymphoblastic Leukemia Therapy on Spermatogonia Populations and Future Fertility

Isolation of spermatogonial stem cells before potentially sterilizing cancer therapy, followed by transplantation of these cells into the testis after such treatment, may be an effective approach to prevent infertility among prepubertal boys suffering from acute lymphoblastic leukemia (ALL). A key clinical consideration in this context is the timing of biopsy, if collection of spermatogonia could be delayed from diagnosis to the later phase of leukemia treatment, better patient selection could be offered. The objective of the study was to examine the routine testicular biopsy material collected to detect testicular leukemia to evaluate if treatment for leukemia affects numbers and maturation of the spermatogonia during the prepubertal period. The study involved 28 testicular biopsies from 23 prepubertal boys treated for ALL. Samples were stained immunohistochemically to evaluate the expression of the spermatogonial markers MAGE 4A, OCT4, CD9, and AP2gamma, and of the Sertoli cell marker WT-1. To relate these findings to gonadal function after sexual maturation, the surviving patients were evaluated as adults. Several MAGE 4A-, CD9-, or OCT4-positive spermatogonia were detected in testicular biopsies after standard risk therapy without cyclophosphamide, whereas their numbers were significantly reduced in six patients receiving high-risk ALL therapy involving cyclophosphamide. No significant alteration in spermatogonial numbers was associated with testicular leukemia. All patients not treated with cyclophosphamide recovered normal testicular function, with normal sperm quality and endocrine function. Treatment for childhood leukemia without high-dose cyclophosphamide seldom depletes the spermatogonial stem cell pool totally.

Body Mass Index and Body Composition in Adolescents Treated with Gonadotropin-Releasing Hormone Analogue Triptorelin Depot for Central Precocious Puberty: Data at Near Final Height

Background/Aim: In children with central precocious puberty (CPP), gonadotropin-releasing hormone (GnRH) analogue treatment has been associated with an increase in body mass index (BMI). We evaluated BMI and body composition in adolescents treated with GnRH analogue at their near final height to assess the long-term effects of therapy on these parameters. Patients and Methods: We studied 20 patients (14.8 ± 1.6 years; 17 females) previously treated with triptorelin depot for CPP (3.75 mg/28 days) from 8.1 ± 0.8 to 11.5 ± 0.8 years. 23 healthy adolescents with normal onset of puberty (14.7 ± 2.1 years, 19 females) were the controls. BMI and body composition (dual-energy x-ray absorptiometry) were assessed. Results: Patients reached their near adult height (–0.5 ± 1.1 standard deviation score (SDS)); the girls were menstruating and the majority (15/17) had regular cycles, the boys showed normal testicular function. BMI was unchanged from the start of GnRH analogue therapy (0.4 ± 1.0 SDS) to near adult height (0.2 ± 1.0 SDS, p = NS vs. 0). Total fat mass (TFM) was significantly increased (16,144 ± 8,065 g; controls 10,712.1 ± 4,120.4 g, p < 0.02); glucose homeostasis and lipid profile corresponded to reference ranges. Conclusions: GnRH analogue therapy did not show long-term detrimental effects on BMI, but it may increase TFM, suggesting that body composition should be monitored till adulthood.

Effects of FSH on testicular mRNA transcript levels in the hypogonadal mouse.

FSH acts through the Sertoli cell to ensure normal testicular development and function. To identify transcriptional mechanisms through which FSH acts in the testis, we have treated gonadotrophin-deficient hypogonadal (hpg) mice with recombinant FSH and measured changes in testicular transcript levels using microarrays and real-time PCR 12, 24 and 72 h after the start of treatment. Approximately 400 transcripts were significantly altered at each time point by FSH treatment. At 12 h, there was a clear increase in the levels of a number of known Sertoli cell transcripts (e.g. Fabp5, Lgals1, Tesc, Scara5, Aqp5). Additionally, levels of Leydig cell transcripts were also markedly increased (e.g. Ren1, Cyp17a1, Akr1b7, Star, Nr4a1). This was associated with a small but significant rise in testosterone at 24 and 72 h. At 24 h, androgen-dependent Sertoli cell transcripts were up-regulated (e.g. Rhox5, Drd4, Spinlw1, Tubb3 and Tsx) and this trend continued up to 72 h. By contrast with the somatic cells, only five germ cell transcripts (Dkkl1, Hdc, Pou5f1, Zfp541 and 1700021K02Rik) were altered by FSH within the time-course of the experiment. Analysis of canonical pathways showed that FSH induced a general decline in transcripts related to formation and regulation of tight junctions. Results show that FSH acts directly and indirectly to induce rapid changes in Sertoli cell and Leydig cell transcript levels in the hpg mouse but that effects on germ cell development must occur over a longer time-span.

Finding of a bull with Y;17 translocation

A bull with a Y;17 translocation was found. This finding was examined by G banding, C banding, and studying the cytodensitometric profiles of G banding. The subject appeared phenotypically normal, with normal reproductive organs and testicular function (GnRH Test). There was a slight pathospermia (oligozoospermia and asthenozoospermia), therefore the portions of Y chromosome with TDF and AZF were not lost.

Androgenic potentials of aqueous extract of Massularia acuminata (G. Don) Bullock ex Hoyl. stem in male Wistar rats

The use of medicinal plants in the management of several ailments is gaining popularity nowadays. Massularia acuminata, one of such plants is commonly used as chewing sticks due to its antimicrobial activity and the aqueous extract of its stem as an aphrodisiac. Aphrodisiac activity in some plants may be due to androgen increasing property of its phytochemicals. Aim of the study This study therefore sought to assess the androgenic potentials of aqueous extract of Massularia acuminata stem in male rats for 21 days. Materials and methods Male rats weighing between 220 and 260 g were completely randomized into four groups: A, B, C and D. Group A, the control received orally 1 ml of distilled water (the vehicle) while groups B, C and D were orally administered with 1 ml each corresponding to 250, 500 and 1000 mg/kg body weight of the plant extract, respectively for 21 days. Rats were sacrificed 24 h after 1, 7 and 21 days. Results Compared with the control, extract administration at all the doses produced significant increase ( P < 0.05) in testes–body weight ratio, testicular protein, glycogen, sialic acid, cholesterol, testosterone, luteinizing and follicle stimulating hormone concentrations throughout the period of administration. Testicular gamma glutamyl transferase activities were decreased significantly ( P < 0.05) after the first dose and was sustained throughout the experimental period. Conclusion The available evidence in this study suggests that aqueous extract of Massularia acuminata stem has androgenic potential which may stimulate male sexual maturation and enhance normal testicular function.

Regulation of testicular function in the stallion: An intricate network of endocrine, paracrine and autocrine systems

It is well established in many mammalian species, including the horse that normal testicular function is dependent upon a functional hypothalamic–pituitary–testicular (HPT) axis, which involves classic feedback mechanisms. The major HPT hormones involved in the stallion are gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), estrogens (Es) and inhibin (INH). Although prolactin (PRL) fluctuates with season in the stallion and both PRL and thyroid hormone (TH) affect reproduction in other male species, their effects on stallion reproduction have not been elucidated. Growth hormone (GH) in the stallion may be involved in sperm motility, production and secretion of insulin-like growth factor-1 (IGF-1) and LH-induced testosterone release. The action of these hormones and the products involved for normal spermatogenesis require cell to cell communication within the testis. The somatic cell types, Leydig, Sertoli and peritubular myoid cells, all support germ cell development, maturation and release into the seminiferous tubule lumen. The cell to cell crosstalk involves an intricate network of paracrine–autocrine systems that support the endocrine input to modulate cell function. In other male species, researchers have demonstrated the reproductive effects of such paracrine–autocrine factors as IGF-1, transferrin, androgens, estrogens, inhibin, insulin like peptide 3 (INSL3), beta-endorphin and oxytocin. The specific nature and relative contribution of these various factors on testicular function in fertile and subfertile stallions are under investigation. This review summarizes current information regarding the nature of the multiple endocrine–paracrine–autocrine systems that may be necessary for normal testicular function in the stallion.

Hormonal regulation of spermatogenesis and spermiogenesis

Normal testicular function is dependent upon hormones acting through endocrine and paracrine pathways both in vivo and in vitro. Sertoli cells provide factors necessary for the successful progression of spermatogonia into spermatozoa. Sertoli cells have receptors for follicle stimulating hormone (FSH) and testosterone which are the main hormonal regulators of spermatogenesis. Hormones such as testosterone, FSH and luteinizing hormone (LH) are known to influence the germ cell fate. Their removal induces germ cell apoptosis. Proteins of the Bcl-2 family provide one signaling pathway which appears to be essential for male germ cell homeostasis. In addition to paracrine signals, germ cells also depend upon signals derived from Sertoli by direct membrane contact. Somatostatin is a regulatory peptide playing a role in the regulation of the proliferation of the male gametes. Activin A, follistatin and FSH play a role in germ cell maturation during the period when gonocytes resume mitosis to form the spermatogonial stem cells and differentiating germ cell populations. In vitro cultures systems have provided evidence that spermatogonia in advance stage of differentiation have specific regulatory mechanisms that control their fate. This review article provides an overview of the literature concerning the hormonal pathways regulating spermatogenesis.

Relationship between testicular volume and testicular function: comparison of the Prader orchidometric and ultrasonographic measurements in patients with infertility

To evaluate the relationship between testicular function and testicular volume measured by using Prader orchidometry and ultrasonography (US) to determine the critical testicular volume indicating normal testicular function by each method. Total testicular volume (right plus left testicular volume) was measured in 794 testes in 397 men with infertility (mean age, 35.6 years) using a Prader orchidometer and also by ultrasonography. Ultrasonographic testicular volumes were calculated as length X width X height X 0.71. To evaluate volume-function relationships, patients were divided into 10 groups representing 5-mL increments of total testicular volume by each method from below 10 mL to 50 mL or more. Mean total testicular volume based on Prader orchidometry and US were 36.8 mL and 26.3 mL, respectively. Semen volume, sperm density, total sperm count, total motile sperm count, and serum FSH, LH, and testosterone all correlated significantly with total testicular volume measured by either method. Mean sperm density was in the oligozoospermic range in patients with total testicular volume below 35 mL by orchidometry or below 20 mL by ultrasonography. Mean total sperm count was subnormal in patients with total testicular volume below 30 mL by orchidometry or under 20 mL by ultrasonography. Testicular volume measured by either ultrasonography or Prader orchidometry correlated significantly with testicular function. However, critical total testicular volume indicating normal or nearly normal testicular function was 30 mL to 35 mL using Prader orchidometer and 20 mL using ultrasonography. Prader orchidometry morphometrically and functionally overestimated the testicular volume in comparison to US.

Long‐term follow‐up and analysis of monozygotic twins concordant for 45,X/46,XY peripheral blood karyotype but discordant for phenotypic sex

We report on the follow-up of a set of monozygotic (MZ) twins who were concordant for peripheral blood karyotype 45,X/46,XY but discordant for phenotypic sex. One twin is a phenotypically normal male and the other twin has asymetrical gonadal dysgenesis. The female twin has the mos45,X/46,XY karyotype in all four tissues: left testis, right streak, vas deferens, and clitoral skin. The normal male twin has the normal 46,XY karyotype in all three tissues tested: foreskin, scrotal skin, and testis. Follow-up of the twins at age 21, revealed persistence of mos45,X/46,XY karyotype in peripheral blood into adult life. However, the male grew up with normal male stature, reaching an adult height of 182 cm. The female twin received low dose estrogen replacement with complete breast development at age 14 years. She reached an adult height of 156 cm. At 21 years of age the male twin had normal testicular endocrine function, but severe oligospermia. The long-term follow-up of this set of MZ twins indicate that the male twin has the mosaicism confined to peripheral blood and has the normal 46,XY male constitution. This was further confirmed by his normal male stature and normal testicular endocrine function. The 45X cell line is likely due to his receiving these cells passively from his twin sister via placental anastomoses in utero. The exposure to these 45,X cells during development may have had an impact on his spermatogenesis.

Constitutive Expression of Prostaglandin-Endoperoxide Synthase 2 by Somatic and Spermatogenic Cells Is Responsible for Prostaglandin E2 Production in the Adult Rat Testis1

Prostaglandins (PGs), particularly PGE(2), have been implicated in the control of testicular steroidogenesis, spermatogenesis, and local immunity. However, virtually nothing is known about the expression or activity of the prostaglandin-endoperoxide synthases (PTGSs; also referred to as the cyclooxygenases), the specific rate-limiting enzymes responsible for PG production, in the adult testis. This activity was investigated in rats under normal conditions and during lipopolysaccharide-induced inflammation using quantitative real-time PCR, in situ hybridization, Western blotting, and PGE(2) measurements by ELISA. The mRNA for both the "constitutive" Ptgs1 and the "inducible" Ptgs2 forms was detected in multiple testicular cell types. Testicular Ptgs2 expression was substantially higher than that of Ptgs1, and testicular production of PGE(2) in vitro was found to be suppressed by a specific PTGS2 inhibitor (NS-398), but not by an inhibitor of PTGS1. Further investigation indicated that 1) PGE(2) production in the adult testis is attributable to constitutive expression of PTGS2 by somatic (Leydig cells and Sertoli cells) and spermatogenic cells; 2) testicular macrophages constitutively produce relatively low levels of PTGS2 and PGE(2) but are the only cell type to respond significantly to an inflammatory stimulus by increasing production of PGE(2); and 3) testicular PTGS2 expression and intratesticular PGE(2) levels are only marginally affected by acute inflammation. These data point toward a previously unanticipated maintenance role for the "inducible" PTGS2 enzyme in normal testicular function, as well as an anomalous response of testicular PTGS2 to inflammatory stimuli. Both observations are consistent with the reduced capacity of the testis to initiate and support inflammatory reactions.

The potential impact of novel investigational compounds on human fertility

There is considerable concern that the incidence of infertility in humans may be increasing, in some instances due to the action of bioactive xenobiotic compounds found in our environment; for example, high concentrations of xenobiotics with estrogenic activity can interfere with normal testicular function and fertility. However, recent studies have shown that very low concentrations of several estrogenic xenobiotics can have subtle, unexpected effects on sperm function. When tested in vitro, these compounds stimulate spermatozoa to become fertile very quickly, but continued stimulation causes them to burn out and lose fertilising ability; similar responses occurring in vivo could reduce fertility. In contrast, several other compounds, structurally related to amfetamine, have been shown to act on spermatozoa in vitro in a positive manner, stimulating cells to ‘switch on’ quickly and then preventing burnout so that they maintain fertilising potential; similar responses occurring in vivo could enhance fertility. These results could have implications for either reducing or enhancing natural fertility.

Obesity and Male Reproductive Potential

Obesity and Male Reproductive Potential

The impact and potential etiology of teratospermia in the domestic cat and its wild relatives

Teratospermia (production of >60% morphologically abnormal sperm/ejaculate) is relatively common among various species in the family Felidae, which is comprised of 37 species. Over two decades of research in this area have produced a significant understanding of the phenotypic expression, its impacts on sperm function and etiology. There is good evidence suggesting that a reduction in genetic diversity contributes to this phenomenon. Results to date demonstrate that spermatozoa from teratospermic donors are compromised in the ability to undergo capacitation and the acrosome reaction, penetrate the zona-pellucida, fertilize conspecific oocytes and survive cryopreservation. Recent studies also reveal abnormalities in chromatin integrity in sperm from teratospermic donors, which, interestingly, fails to impact fertilization or embryo development after intracytoplasmic sperm injection. Through planned inbreeding studies, we now have established that teratospermic cats also produce more spermatozoa by virtue of more sperm producing tissue, more germ cells per Sertoli cell and reduced germ cell loss during spermatogenesis. Overall, it now is clear that gain in sperm quantity is achieved at the expense of sperm quality, suggesting an extensive disruption of normal testicular function in teratospermic donors. Preliminary studies on testicular gene expression in teratospermic cats have also revealed abnormal expression patterns. These findings have markedly increased our understanding of testis biology in the teratospermic donor and reaffirm the value of cats, including wild species, as models for studying novel regulatory mechanisms controlling spermatogenesis and spermiogenesis.

Laminin α1 Chain Corrects Male Infertility Caused by Absence of Laminin α2 Chain

Laminins are important for basement membrane structure and function. The laminin alpha2 chain is a major component of muscle basement membranes, and mutations in the laminin alpha2 gene lead to congenital muscular dystrophy in humans and mice. Although the laminin alpha2 chain is prominently expressed in testicular basement membranes, its role in testis has remained unclear. Here, we show that laminin alpha1, alpha2, beta1, beta2, gamma 1, and gamma 3 chains are the major laminin chains in basement membranes of seminiferous tubules. In laminin alpha2 chain-deficient dy(3 K)/dy(3 ASK) mice, lack of laminin alpha2 chain led to concurrent reduction of laminin gamma 3 chain and abnormal testicular basement membranes. Seminiferous tubules of laminin alpha2 chain-deficient dy(3 K)/dy(3 K) mice displayed a defect in the timing of lumen formation, resulting in production of fewer spermatides. We also demonstrate that overexpression of laminin alpha1 chain in testis of dy(3 K)/dy(3 K) mice compensated for laminin alpha2 chain deficiency and significantly reversed the appearance of the histopathological features. We thus provide genetic data that laminin alpha chains are essential for normal testicular function in vivo.

PRL Secreting Adenomas in Male Patients

Prolactinomas are the most frequent pituitary tumors and their frequency varies with age and sex, occurring most frequently in females between 20-50 yr-old. In men, hyperprolactinaemia is often present for many years without symptoms, as generally the most important symptoms are the decrease in libido and/or sexual potency both underestimated by the majority of the patients. Prolactin (PRL) plays a role in the process of spermatogenesis, and normal serum PRL levels are required for normal testicular function. On the other hand, hyperprolactinaemia has multiple negative effects on the gonadal axis. As a consequences hyperprolactinemic males show alteration of sexual potency and seminal fluid quality. Cabergoline treatments is able to induce normalization of PRL levels and a reduction of tumor mass in the majority of patients and consequently restoring the normal semen quality and ameliorating the quality of life of men with pituitary PRL-secreting adenoma.

028. Cytokine networks and regulation of spermatogenesis - what should we really believe?

Spermatogenesis is a complex yet highly organised process involving intimate interactions between the supporting Sertoli cells and germ cells at various stages of development. The repeating pattern of the cycle of the seminiferous epithelium is due to the fact that spermatogonia enter spermatogenesis at regularly spaced intervals and proceed through the process at a species-specific rate. How this degree of coordination is maintained remains poorly understood, but recent evidence has focussed attention on the role of growth factors produced by the Sertoli cells and germ cells. Several of these growth factors, such as interleukin-1α (IL-1α), IL-6, tumour necrosis factor (TNFα) and activin A, are also inflammatory cytokines. This has led some researchers to question the physiological significance of these data with respect to normal testicular function. For example, in spite of the fact that IL-1α is produced by the Sertoli cell and regulates spermatogonial proliferation and development in vitro, mice lacking the IL-1R, and hence unresponsive to IL-1α, possess relatively normal fertility. So what role, if any, do these cytokines play in the normal testis, or are they only important during inflammation? It is quite evident that these cytokines have stimulatory and/or inhibitory effects on spermatogonial and spermatocyte development. These cytokines also interact at multiple levels within each other’s signalling pathways and have considerable redundancy of action. Moreover, expression of these cytokines varies across the cycle of the seminiferous epithelium, with major changes in production coinciding with two key events within the cycle: the release of sperm from the epithelium, and the major peaks of DNA synthesis by spermatogonia and preleptotene spermatocytes. It is therefore possible to hypothesise that release of sperm and resorption of the residual cytoplasm triggers a self-regulating inflammatory cascade within the epithelium that initiates and then modulates the next round of spermatogenic development, ensuring that spermatogonia enter the process at the appropriately spaced intervals.

Germinal tumor invasion and the role of the testicular stroma.

Testicular germ cell tumors (TGCTs) are the most frequent neoplasia among young people and their incidence has grown very quickly during recent decades in North America and Europe. Many studies have been carried out in order to elucidate the factors involved in the appearance and progression of these tumors. Little is known about the role of cancer cell-stroma crosstalk in TGCT invasive processes. Here, we review several factors which may be implicated in germ cell tumor progression, such as matrix metalloproteinases, insulin-like growth factor, transforming growth factor beta, the cadherin/catenin complex and integrins. Paradoxically, some of these molecules are also involved in the regulation of normal testicular function. Finally, we discuss prospects for future research on the role of the stroma in the progression and differentiation of male germ cell tumors.

Isolation and expression analysis of the testis-specific gene, STRA8, stimulated by retinoic acid gene 8.

Retinoids are known to be required for vertebrate reproduction, and in the male, for the maintenance of normal testicular structure and function. Previously several novel retinoic acid responsive genes, collectively designated as the Stra genes, had been isolated in the mouse. The Stra8 gene encodes a cytoplasmic protein and is expressed specific to the developing male gonad during mouse embryogenesis. In adult mouse, its expression is restricted to the premeiotic germ cells. Thus it has been suggested that the mouse Stra8 protein may play a role in the premeiotic phase of spermatogenesis. Recently a lot of genes that are expressed only in male germ cells have been isolated in the mouse. The mouse Stra8, Rnh2, Piwil2, Tex17, and Tuba7 were identified as testis-specific expressed genes. In addition, the Figla was known to be a testis- and ovary-specific gene. Recently we had reported the isolation of the human RNH2 cDNA and its expression, which is limited to the human testis. In the present study, we have isolated full-length cDNA of S TRA8 and partial cDNAs of PIWIL2, FIGLA, TEX17, and TUBA7, and analyzed their expression patterns in human tissues.