Rosália Sá

Effect of insulin deprivation on metabolism and metabolism-associated gene transcript levels of in vitro cultured human Sertoli cells.

BACKGROUND Sertoli cells metabolize glucose producing lactate for developing germ cells. As insulin regulates glucose uptake and its disturbance/insensitivity is associated with diabetes mellitus, we aimed to determine the effect of insulin deprivation in human Sertoli cell (hSC) metabolism and metabolism-associated gene expression. METHODS hSC-enriched primary cultures were maintained in the absence/presence of insulin and metabolite variations were determined by (1)H-NMR. mRNA expression levels of glucose transporters (GLUT1, GLUT3), lactate dehydrogenase (LDHA) and monocarboxylate transporter (MCT4) were determined by RT-PCR. RESULTS Insulin deprivation resulted in decreased lactate production and in decrease of glucose consumption that was completely reverted after 6h. Cells of both groups consumed similar amounts of glucose. In insulin-deprived cells, transcript levels of genes associated to lactate metabolism (LDHA and MCT4) were decreased. Transcript levels of genes involved in glucose uptake exhibited a divergent variation: GLUT3 levels were decreased while GLUT1 levels increased. Insulin-deprived hSCs presented: 1) altered glucose consumption and lactate secretion; 2) altered expression of metabolism-associated genes involved in lactate production and export; 3) an adaptation of glucose uptake by modulating the expression of GLUT1 and GLUT3. GENERAL SIGNIFICANCE This is the first report regarding the effect of insulin-deprivation on hSC metabolism.

Influence of 5α-dihydrotestosterone and 17β-estradiol on human Sertoli cells metabolism

Sertoli cells metabolize glucose, converting it to lactate that is used by developing germ cells for their energy metabolism. Androgens and oestrogens have metabolic roles that reach far beyond reproductive processes. So, the main purpose of this study was to examine the effect of sex steroid hormones on metabolite secretion/consumption in human Sertoli cells. Human Sertoli cell-enriched primary cultures were maintained in a defined medium for 50 h and glucose, pyruvate, lactate and alanine variations were determined using (1) H-NMR spectra analysis, in the absence or presence of 100 nm 17β-estradiol (E(2) ) or 100 nm 5α-dihydrotestosterone (DHT). The mRNA expression levels of glucose transporters, lactate dehydrogenase and monocarboxylate transporters were also determined using semi-quantitative RT-PCR. Cells cultured in the absence (control) or presence of E(2) consumed the same amounts of glucose at similar rates during the 50 h. During the first 15 h of treatment with DHT, glucose consumption and glucose consumption rate were significantly higher. Nevertheless, DHT-treated cells secreted a significantly lower amount of lactate than control and E(2) -treated cells. Such a decrease was concomitant with a significant decrease in lactate dehydrogenase A mRNA levels after 50 h treatment in DHT-treated groups. Finally, alanine production was significantly increased in E(2) -treated cells after 25 h treatment, which indicated a lower redox/higher oxidative state for the cells on those conditions. These results support the existence of a relationship between sex steroid hormones action and energy metabolism, providing the first assessment of androgens and oestrogens as metabolic modulators of human Sertoli cells.

Ultrastructure of tubular smooth endoplasmic reticulum aggregates in human metaphase II oocytes and clinical implications.

OBJECTIVE To compare demographic, embryologic, pregnancy, and newborn outcomes after intracytoplasmic sperm injection (ICSI) cycles with or without mature oocytes (metaphase II [MII]) showing visible aggregates of tubular smooth endoplasmic reticulum (aSERT) and to describe the ultrastructure of this dysmorphism. DESIGN Retrospective study. SETTING Private fertility center and university cell biology and genetics departments. PATIENT(S) There were 721 ICSI cycles, 520 carrying morphologically normal MII (control group) and 60 containing aSERT-MII (study group). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Embryologic and clinical and live birth outcomes, including malformations and ultrastructural characterization of aSERT-MII. RESULT(S) Compared with the control group there was a significant decrease in the fertilization, embryo cleavage, and blastocyst rates in the study group. The only child born after transfer of embryos derived from aSERT-MII presented a major cardiovascular malformation. Ultrastructurally, large aSERT were surrounded by abnormal-shaped mitochondria and clusters of small dense bodies formed by very small vesicles, and they had curvilinear dense tubules in the interior. The same pathology was observed in small peripheral aSERT. CONCLUSION(S) The presence of large aSERT, showing attainment of the periphery, demonstrated that the cytoplasm is pathologic. The compromised embryo development and implantation was associated with decreased clinical outcomes and newborn malformations. Therefore, oocytes with large aSERT should not be used for embryo transfer.

Alternatively Spliced Protein Variants as Potential Therapeutic Targets for Male Infertility and Contraception

Abstract: Mammalian sperm were previously shown to express the PP1γ2 isoform of protein phosphatase 1 (PP1) as well as its regulatory proteins inhibitor 2 and glycogen synthase kinase 3. Furthermore, the development of sperm motility during transit through the epididymis correlates with changes in PP1 activity. Thus, since PP1 cellular activity is determined by the partners it binds, we embarked on a study aimed at defining the specific interactomes of PP1γ1 and PP1γ2 (the two known alternatively spliced variants of PP1γ). To this end, exhaustive screens were performed on a human testis cDNA library using the yeast two‐hybrid method. Among the various proteins detected, the most abundant interactors with PP1γ2 were Nek2A and R15B. Closer sequence analysis revealed novel alternatively spliced variants of Nek2A and NIPP1, which we designated Nek2A‐T and NIPP1‐T, respectively. They were shown to be highly expressed in rat and human testis by Northern analysis and to result from alternative splicing events by RT‐PCR. Thus, both the previously known Nek2A isoform and the novel Nek2A‐T and NIPP1‐T variants appear to bind PP1γ2 in vitro (blot overlays) and in vivo by coexpression in yeast. The usefulness of testis‐specific alternatively spliced proteins as targets for the development of novel therapeutic strategies for male infertility and contraception is discussed. PP1γ2, Nek2A‐T, and NIPP1‐T are currently being investigated as alternatively spliced targets for signal transduction therapeutics.

Cytological and Expression Studies and Quantitative Analysis of the Temporal and Stage-Specific Effects of Follicle-Stimulating Hormone and Testosterone During Cocultures of the Normal Human Seminiferous Epithelium

Abstract In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.

Molecular characterization of the cystic fibrosis transmembrane conductance regulator gene in congenital absence of the vas deferens

Purpose: Approximately 20% of patients with congenital absence of the vas deferens remain without two mutations identified. We applied a strategy of serial screening steps to 45 patients with congenital absence of the vas deferens and characterized cystic fibrosis transmembrane conductance regulator gene mutations in all cases.Methods: DNA samples of 45 patients with congenital absence of the vas deferens were screened by successive different molecular genetics approaches.Results: Initial screening for the 31 most frequent cystic fibrosis mutations, IVS8 poly(TG)m, poly(T)n, and M470V polymorphisms, identified 8 different mutations in 40 patients (88.9%). Extensive cystic fibrosis transmembrane conductance regulator gene analysis by denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography, and DNA sequencing detected 17 further mutations, of which three were novel. Cystic fibrosis transmembrane conductance regulator gene rearrangements were searched by semiquantitative fluorescent multiplex polymerase chain reaction, which detected a CFTRdele2,3 (21 kb) large deletion and confirmed two homozygous mutations. Overall, 42 patients (93.3%) had two mutations and 3 patients (6.7%) had one mutation detected.Conclusions: The present screening strategy allowed a higher mutation detection rate than previous studies, with at least one cystic fibrosis transmembrane conductance regulator gene mutation found in all patients with congenital absence of the vas deferens.

Major regulatory mechanisms involved in sperm motility.

The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies.

Physiology of Na+/H+ Exchangers in the Male Reproductive Tract: Relevance for Male Fertility

ABSTRACT The maintenance of pH homeostasis in the male reproductive tract is kept through the involvement of several mechanisms, among which is included the transmembranous movement of H+ ions. Na+-H+ exchangers (SLC9, solute carrier 9 family members) are among the membrane transporters known to participate in intracellular and extracellular pH regulation but also have important roles in salt and water absorption across epithelia and in the regulation of cell volume. The presence of several Na+-H+ exchangers has been reported in the male reproductive tract. Their involvement in the processes that ensure the correct pursuance of the spermatogenetic event and spermatozoa maturation has been suggested. Indeed, the formation of mature spermatozoa is highly dependent on the maintenance of adequate ductal luminal milieu pH and ionic balance. Perturbations in these processes result in reduced male reproductive potential and consequently male subfertility and/or infertility. Thus, it is imperative to understand H+ transport dynamics in order to identify and counteract possible alterations associated with reduced male fertility caused by pathological conditions. Herein, we will discuss the expression pattern and physiological roles of SLC9 family members in the cells of the male reproductive tract as well as the molecular basis of H+ transport and its involvement in male reproductive potential.

Aquaporin‐9 is expressed in rat Sertoli cells and interacts with the cystic fibrosis transmembrane conductance regulator

Men with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are usually subfertile/infertile. Besides playing a role in Cl−/HCO3− transport, it has been proposed that CFTR interacts with water membrane transport systems, particularly aquaporins, to control seminiferous tubular secretion, which is regulated by the somatic Sertoli cells (SCs). As aquaporin‐9 (AQP9) is highly expressed throughout the male reproductive tract, we hypothesized that it is also present in rat SCs and that it physically interacts with CFTR. To test this hypothesis, primary cultures of rat SCs were established, and expression of CFTR and AQP9 was assessed by RT‐polymerase chain reactions (mRNA) and Western blot analysis (protein). A coimmunoprecipitation assay was used to evaluate the physical interaction between CFTR and AQP9. Our results show that CFTR and AQP9 are expressed in rat SCs. We were also able to detect a molecular interaction between CFTR and AQP9 in rat SCs. This is the first report describing the presence of AQP9, and its interaction with CFTR, in rat SCs. Moreover, our results provide evidence that CFTR is involved in water homeostasis of the seminiferous tubular secretion. These mechanisms may open new insights on therapeutic targets to counteract subfertility/infertility in men with cystic fibrosis and mutations in the CFTR gene.

Cryopreservation of human testicular diploid germ cell suspensions.

For patients with threatened fertility, preservation of it is a major concern. Although promising results have been obtained in animal models using testicular germ cell suspensions, in humans, it is crucial to first develop an efficient method of cryopreservation to be able to apply to transplantation. Thus, four reliable and available cryopreservation techniques in any fertility centre were tested to cryopreserve an enriched fraction of diploid germ cells isolated from human testicular biopsies. The protocols were evaluated based on cell viability, and the results showed significant differences between the four methods. The semen and tissue cryopreservation methods appeared to be inadequate for diploid germ cell suspensions, and programmed slow freezing gave significantly lower results than open pulled straw vitrification; the latter was found to be the protocol that best preserved cell viability. The vitrification of isolated human diploid germ cells is innovative and constitutes valuable information for cryopreservation in cases of transplants or in vitro maturation.