Asthenozoospermia and membrane remodeling enzymes: a new role for phospholipase A2.

Phosholipase A2 (PLA2 ) activity in the seminal plasma and in sperm heads is closely related to sperm motility and male fertility. Therefore, the purpose of this study was to investigate the possible involvement of different isoforms of phospholipase in asthenozoospermia. To accomplish this, cPLA2 , phospho-cPLA2 , iPLA2 , and sPLA2 were evaluated by immunofluorescence and immunoblot analyses in spermatozoa obtained from 22 normozoospermic men and 28 asthenozoospermic patients. We found significant differences in cPLA2 and its phosphorylated/activated form, iPLA2 , and sPLA2 content and distribution in normal and asthenozoospermic patients. cPLA2 was localized in heads, midpieces, and tails of all spermatozoa as constitutive enzyme, less expressed in the tail of spermatozoa with low progressive motility. While active phospho-cPLA2 distribution was homogeneous throughout the cell body of control-donor spermatozoa, lower levels were detected in the tails of asthenozoospermic patients, as opposed to its strong presence in heads. Low immunofluorescence signal for iPLA2 was found in astenozoospermic patients, whereas sPLA2 was significantly lower in the heads of asthenozoospermic patients. Spermatozoa with low progressive motility showed differences both in terms of total specific activity and of intracellular distribution. cPLA2 , iPLA2 , and sPLA2 specific activities correlated positively and in a significantly manner with sperm progressive motility both in normozoospermic men and asthenozoospermic patients. In conclusion, PLA2 s are expressed in different areas of human spermatozoa. Spermatozoa with low motility showed differences in total specific activity and enzyme distributions. We speculated that PLA2 expression and/or different distribution could be potential biomarkers of asthenozoospermia, one of the major causes of male factor infertility.

Effects of anti-neoplastic treatment on sperm aneuploidy rate in patients with testicular tumor: a longitudinal study.

OBJECTIVE: The adjuvant radio/chemotherapy, usually employed after orchidectomy in patients with testicular tumors, allows a long-term survival with a consequent increased request for fertility. However, little is known about the effects of the anti-neoplastic treatment on sperm cytogenetic asset. Therefore, this prospective, longitudinal study was designed to evaluate the effects of radio- and/or chemotherapy on sperm chromosome. METHODS: Eleven patients with testicular tumor were enrolled and underwent sperm aneuploidy rate evaluation before and after 3, 6, 9, 12, 18, 24, and 36 months from radio- and/or chemo-therapy ending. A double and triple multicolor fluorescence in-situ hybridizations for chromosomes 8, 12, 18, X and Y were used to evaluate the sperm aneuploidy rate. To define normal sperm aneuploidy rate, 18 healthy, normozoospermic men were selected as controls. RESULTS: Before treatment, testicular tumor patients had a higher total sperm aneuploidy rate compared with normal men. Total sperm aneuploidy rate showed a slight, but statistically significant increase 6 months after anti-neoplastic treatment. This increase was mainly related to the high sperm aneuploidy rate found in 2 patients which remained elevated up to 12 months in both of them. CONCLUSION: These results showed that anti-neoplastic treatment caused only slight and transient sperm malsegregation events in patients with testicular tumor. However, since a subset of them had an elevated sperm aneuploidy rate for about 1 yr, we suggest to counsel them to refrain from fatherhood for this length of time.

Chromosome abnormalities in spermatozoa of patients with azoospermia and normal somatic karyotype.

Since epididymal and testicular spermatozoa of azoospermic patients are frequently used for intracytoplasmic sperm injection (ICSI), many studies have been carried out to evaluate their karyotype. This article will review all published data on this topic. In most of the studies, spermatozoa have been retrieved from the testis or the epididymis of patients with nonobstructive (NOA) or obstructive (OA) azoospermia, respectively. Sperm aneuploidy has been evaluated by fluorescence in situ hybridization using probes for sex chromosomes and an array of autosomes. A significantly higher sperm aneuploidy rate has been reported in patients with NOA and OA compared to ejaculated spermatozoa, mainly for sex chromosomes. The magnitude of the increase varies between studies, probably because of the heterogeneity of case selection as well as of the methodology employed. The majority of the studies reported that patients with NOA have a greater sperm aneuploidy rate compared to OA. The greater frequency of sperm aneuploidy in azoospermic patients increases the risk of transmitting a karyotype abnormality to the offspring generated by ICSI.

Sperm aneuploidy in infertile men.

A recent line of research has shown that infertile male patients produce cytogenetically abnormal spermatozoa, despite a normal somatic karyotype, as a result of an altered intra-testicular environment that affects negatively the mechanisms controlling chromosome segregation during cell division. The rate of aneuploid spermatozoa production is significantly higher in patients with abnormal sperm parameters compared with those of normozoospermic subjects or infertile patients with normal sperm parameters. All chromosomes are subject to aneuploidy, although at a different rate; the heterochromosomes are more often altered than are the autosomes. A negative correlation has been reported to exist between aneuploidy and the main sperm parameters, suggesting that greater testicular damage is associated with a greater chance of chromosome malsegregation events. Abnormally-shaped spermatozoa are more likely to have chromosome abnormalities, particularly those with an enlarged head. More studies are necessary, however, to evaluate whether other types of sperm head abnormalities are also associated with an abnormal sperm chromosome complement. The possibility of retrieving testicular or epididymal spermatozoa in patients with azoospermia and using them in assisted reproduction techniques has prompted the evaluation of their chromosomal status. Studies have shown that testicular and epididymal spermatozoa have a greater rate of aneuploidy compared with that of ejaculated spermatozoa. Some authors have also shown that patients with non-obstructive azoospermia have a significantly higher sperm aneuploidy rate compared with that of patients with obstructive azoospermia. Sperm aneuploidy seems to have a negative impact on assisted reproduction technique outcome. Although it does not affect the fertilization rate, an elevated sperm aneuploidy rate is associated with a greater rate of pregnancy failure. Nevertheless, some patients with elevated sperm aneuploidy rate can still achieve a pregnancy, but with an increased risk of generating an aneuploid offspring. Thus, sperm aneuploidy evaluation is recommended in infertile patients with abnormal semen parameters, particularly if they undergo IVF programmes.

Corticotrophin-releasing hormone inhibits insulin-like growth factor-I release from primary cultures of rat granulosa cells.

Corticotrophin-releasing hormone (CRH), a neuropeptide which modulates gonadal function during stress, is expressed by several cell types of the rat ovary and is able to suppress oestrogen release from rat granulosa cells. The mechanism of this effect is, however, not known. Since insulin-like growth factor (IGF)-I is produced by rat granulosa cells and exerts a synergistic role with FSH on granulosa cell steroidogenesis, we hypothesised that CRH may suppress oestrogen release from granulosa cells by inhibiting IGF-I release and/or stimulating the release of its binding protein (IGFBP-3). To test this hypothesis, granulosa cells were obtained from immature female Sprague-Dawley rats primed with diethylstilboestrol, and hormone concentrations were measured in the conditioned medium by radioimmunoassay. CRH suppressed oestrogen and IGF-I release stimulated by FSH used at a concentration of 1 IU/l, whereas it did not have any statistically significant effect on oestrogen and IGF-I release in basal conditions or in response to 5 IU/l FSH. The suppressive effects of CRH on oestrogen and IGF-I release were antagonised by a selective CRH receptor antagonist. CRH had no effects on IGFBP-3 release. CRH did not have any effect on oestrogen release stimulated by increasing concentrations of IGF-I and its suppressive effect on FSH-stimulated oestrogen release was overcome by the addition of low doses of exogenous IGF-I. In conclusion, CRH suppressed the release of oestrogen and IGF-I, but not of IGFBP-3. Thus, the inhibitory effects of CRH on oestrogen release could be mediated, partly, by a suppression of the autocrine/paracrine action of IGF-I.

High sperm aneuploidy rate in unselected infertile patients and its relationship with intracytoplasmic sperm injection outcome.

BACKGROUND: Men with oligoasthenoteratozoospermia (OAT) frequently undergo intracytoplasmic sperm injection (ICSI) as a treatment for their infertility. However, there is an increased risk of transmitting chromosomal abnormalities to the offspring given that natural selection is bypassed by the use of this technique and patients have an increased rate of sperm aneuploidy which, in addition, may negatively affect ICSI outcome. For this reason, the rate of sperm aneuploidy in unselected patients undergoing ICSI and its impact on ICSI performance have been evaluated. METHODS: Aneuploidy and diploidy were evaluated in spermatozoa separated by swim-up for oocyte injection, using DNA probes for chromosomes 8, 12, 18, X and Y. RESULTS: ICSI patients had sperm aneuploidy and diploidy rates significantly higher than those of 13 normozoospermic men who served as controls. Although the total aneuploidy rate varied considerably between the 18 patients, 15 of them (83%) had values above the upper range of the control group. Eighteen ICSI cycles were performed with an overall fertilization rate of 95% and a pregnancy rate of 39%. The aneuploidy rate of the 11 patients whose wives did not achieve pregnancy was slightly higher than that of pregnant couples, but the difference did not reach statistical significance. However, 10 patients in this group (91%) had a sperm aneuploidy rate well above the upper limit of the controls as compared with two patients in the "pregnant" group (29%). CONCLUSIONS: This study has shown that unselected patients undergoing ICSI had an elevated sperm aneuploidy rate. Lack of pregnancy was associated with a tendency towards an increased aneuploidy rate; however, pregnancy occurred even in the presence of an elevated sperm aneuploidy rate.

Genetic, andrological and clinical characteristics of patients with congenital bilateral absence of the vas deferens.

The possibility of retrieving spermatozoa from the epididymis allows patients with congenital bilateral absence of the vas deferens (CBAVD) to father a child by means of assisted reproduction techniques. This has, however, increased the chance of transmitting a mutated allele of the cystic fibrosis transmembrane conductance regulator (CFTR) gene which increases the risk of generating offspring with cystic fibrosis (CF). Because of the increased heterogeneity of the CFTR locus, the study of a discrete number of mutations, as usually carried out in a diagnostic work-up, is unable to ascertain the presence of a mutation in a relatively high proportion of the patients screened. In an attempt to increase the chance of detecting the presence of CFTR gene abnormalities, 37 patients with CBAVD and one patient with congenital unilateral agenesis of the vas deferens (CUAVD) underwent an enlarged diagnostic protocol, which included screening for the most expected mutations of the CFTR gene in our population, evaluation of the five thymidine (5T) allelic variant, sweat test, respiratory function tests, evaluation of steatocrit, and an accurate evaluation of the history of the patient to search for symptoms commonly found in patients with CF. A single CFTR gene mutation was found in 18 patients (48.6%) with CBAVD and in the patient with CUAVD. The most frequent mutation observed was the Delta F508. Eleven patients (45.8%) had the 5T variant and in five of them it was not associated with any detectable mutation of the CFTR gene. Two female partners were found to be carriers of a mutation, whereas 5 (18.5%) had the 5T variant. As many as 71% of CBVAD patients had the simultaneous presence of at least two signs and/or symptoms suggestive of CF, albeit they were of mild intensity and the patients felt fit and healthy. In conclusion, these results suggested that some patients with CBAVD without CFTR gene mutation or 5T variant, even when their sweat test is negative, may show clinical suspicion of carrying a CFTR gene mutation and therefore are at risk of generating children affected by CF if the partner carries a mutation as well. The screening for mutations and a careful clinical examination may contribute to better identification of patients with CFTR-related CBAVD.

Concentrations of insulin-like growth factor (IGF)-I and IGF binding protein-3 in the follicular fluid of women undergoing ovarian hyperstimulation with different gonadotropin preparations.

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) have an important regulatory role in follicular development and oocyte maturation. The aim of this prospective, randomized, double-blind study was to measure the concentrations of IGF-I and IGFBP-3 in follicular fluids collected from infertile women undergoing ovarian hyperstimulation using three different gonadotropin preparations. Twenty infertile women (mean age 33 years, range 28-40) undergoing in vitro fertilization (IVF) programs were recruited. After a written informed consent, each woman randomly underwent a long-protocol for ovarian hyperstimulation using gonadotropin-releasing hormone (GnRH)-analog and one of the following recombinant- and urinary-gonadotropins--alpha-follitropin, beta-follitropin or urofollitropin. Serum 17 beta-estradiol (E2) levels and follicle growth were assessed during the follicular phase. The concentrations of IGF-I and IGFBP-3 in the follicular fluid of aspirated dominant follicles were measured directly. Women treated with alpha-follitropin needed significantly lower doses of follicle-stimulating hormone (FSH) compared to those receiving beta-follitropin (p < 0.05). No other statistically significant differences were detected between groups. Serum E2 levels increased in the three groups from early to late follicular phase. Follicular fluid IGF-I and IGFBP-3 concentrations did not differ significantly in the three groups of women. A statistically significant relationship was observed between follicular fluid IGF-I and IGFBP-3 levels (r = 0.41, p = 0.001). Oocyte maturation correlated in a positive manner with IGF-I (r = 0.34, p = 0.01) and IGFBP-3 (r = 0.29, p = 0.03). These findings show that both recombinant- and urinary-gonadotropin preparations were equally effective in releasing IGF-I and IGFBP-3 in the follicular fluid of dominant follicles, and confirmed the role of these compounds on oocyte maturation.

Effects of progesterone on sperm function: mechanisms of action.

Progesterone stimulates sperm functions, e.g. hyperactivation, acrosome reaction, binding to oocyte zona pellucida and penetration rate into the hamster oocyte. The physiological relevance of these effects has been shown using female genital tract fluids which modulate sperm function according to their progesterone content. Progesterone interacts with specific sperm binding sites that, unlike the classic nuclear receptors, are located on the plasma membrane of the spermatozoon. Binding studies have revealed the presence of two classes of progesterone receptors in the human spermatozoon, one class has an elevated affinity constant (nanomolar) and is specific for progesterone, whereas the other class has an affinity constant in the micromolar range and binds equally well other hydroxylated progesterone derivatives. Following exposure to progesterone, the main event is a rapid (within seconds) increase of the intracellular free calcium concentration, followed by a sustained rise lasting for several minutes (plateau phase). Both these calcium transients are dependent upon entry of extracellular calcium. The nature of the calcium channel that mediates the effects of progesterone is, currently, unknown. It has been postulated that it may be: (i) part of the progesterone receptor; (ii) voltage-dependent; or (iii) operated by second messengers following activation of the progesterone receptor. Progesterone also modulates sperm function by stimulating a trypsin-like proteolytic activity, the biosynthesis of polyamine (putrescine and spermidine), phospholipase A2 activity and protein tyrosine kinase activity in the sperm cell. Recent studies have shown that chloride ion efflux is vital for progesterone to promote the acrosome reaction. This effect is achieved by interaction with a sperm membrane receptor which resembles the neuronal GABA(A) receptor. Accordingly, GABA(A) receptors have been found in the spermatozoon plasma membrane and GABA stimulates hyperactivation and promotes the acrosome reaction.

Glucocorticoids inhibit gonadotropin-releasing hormone by acting directly at the hypothalamic level.

Glucocorticoids, the end-product of the hypothalamic-pituitary-adrenal (HPA) axis, suppress gonadotropin release by acting at the level of the pituitary gland. However, experimental evidence suggests that they may also act at the hypothalamic level to suppress gonadotropin-releasing hormone (GnRH) release. The lack of a direct demonstration of this assumption, prompted us to evaluate the effects of glucocorticoids on hypothalamic GnRH release from individually-incubated hemi-hypothalami explanted from male rats. Since testosterone (T), dihydrotestosterone (DHT), and progesterone suppress GnRH release and androgens potentiate the effects of glucocorticoids on GnRH release, we studied also the interaction of these steroids with glucocorticoids on GnRH release. Corticosterone (B), the main glucocorticoid of the rodents with greater affinity for the type I glucocorticoid receptor, and dexamethasone (DEX), a synthetic type II glucocorticoid receptor agonist, were able to suppress basal GnRH release in a concentration-dependent fashion. DEX induced a more profound suppression of GnRH release. Neither T (0.1 nM) nor DHT (0.01 nM) modulated the suppressive effects of low (10 nM) or high (100 nM) concentrations of B on GnRH release. On the other hand, progesterone counteracted the suppressive effect of low concentrations of B (10 nM) on GnRH release, but had no effect on the suppression caused by a higher concentration of B (100 nM). The ability of glucocorticoids to inhibit directly GnRH release suggests that these stress-responsive hormones act also at the hypothalamic level to suppress the reproductive function. The suppressive effect of B was not modulated by androgens, but it was neutralized by progesterone, at least when B was used at low concentrations. We speculate that this steroid "protects" the GnRH-secreting neuron only during basal, but not stress-induced, HPA axis activity when the concentrations of glucocorticoids are more elevated.

Gamma-aminobutyric acid (GABA) A and B receptors mediate the stimulatory effects of GABA on the human sperm acrosome reaction: interaction with progesterone.

OBJECTIVE: To evaluate which gamma-aminobutyric acid (GABA) receptor mediates the stimulatory effects of this neurotransmitter on the human sperm acrosome reaction, and to examine the interaction of progesterone, a physiologic inducer of the acrosome reaction, with the GABA(A) receptor. DESIGN: Prospective study. SETTING: A university clinic of andrology. PATIENT(S): Men with normal sperm analysis parameters. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The acrosome reaction of motile spermatozoa. RESULT(S): The acrosome reaction was stimulated by GABA in a dose-dependent manner. This effect was inhibited completely by bicuculline, a GABA(A) receptor antagonist, and only partially by saclofen, a GABA(B) receptor antagonist. Accordingly, muscimol, a GABA(A) receptor agonist, stimulated the acrosome reaction to the same extent as GABA, whereas baclofen, a GABA(B) receptor agonist, was less effective. Preincubation with progesterone followed by the addition of GABA resulted in a significant increase in the percentage of acrosome-reacted spermatozoa compared with progesterone alone. However, this increase was less than a simple addition of effects, suggesting that GABA and progesterone act through the same receptor and/or use the same mechanism of action. To test this hypothesis, the ability of progesterone to induce acrosome reaction was tested in the presence of bicuculline, which suppressed the stimulatory effects of progesterone. Given that the GABA(A) receptor is linked to the chloride channel, we tested whether picrotoxin, a blocker of this channel, could modulate the effects of progesterone or GABA. Picrotoxin completely suppressed the acrosome reaction induced by progesterone and only partially suppressed that caused by GABA. CONCLUSION(S): gamma-Aminobutyric acid stimulated the acrosome reaction in human spermatozoa, acting mainly through the GABA(A) receptor and to a lesser extent through the GABA(B) receptor. Progesterone interacted with the GABA(A) receptor to induce the acrosome reaction, and the functional integrity of the chloride channel was vital for this effect.

Macrophage-derived cytokines in the follicular fluids of women with infertility due to immunological causes. Elevated levels of interleukin 6 and low levels of granulocyte-macrophage colony-stimulating factor.

The cytokines are (glyco)proteins secreted by lymphoid and non-lymphoid cells which modulate several biological responses including the ovarian function. Interleukin (IL)-1 and tumour necrosis factor (TNF)-alpha suppress 17beta-estradiol (E2) and progesterone release from granulosa and luteal cells in vitro. TNF-alpha affects negatively folliculogenesis and ovarian maturation. Additional in vivo evidence for a role of this macrophage-derived cytokine came from our recent observation that women with infertility due to immunological causes have elevated intrafollicular fluid levels of TNF-alpha and decreased levels of E2 compared to women with a tubal factor of infertility. Because the macrophages are a primary component of the intrafollicular compartment, the present study was undertaken to evaluate whether other macrophage-derived cytokines are also released in the human follicular fluid. To accomplish this, we measured the levels of IL-1beta, IL-6, IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the follicular fluids of two groups of infertile women undergoing to an in vitro fertilization program. The first group of women had a significant titre of anti-spermatozoon antibodies in the serum and/or the mucus as the only cause of infertility, whilst the second group of women was infertile because of non patent fallopian tubes. Intrafollicular cytokines levels were measured by solid-phase ELISA and steroid concentrations by radioimmunoassay. Whilst IL-1beta, IL-6, IL-10, and GLM-CSF were all measurable in the follicular fluid of both groups of women, the levels of IL-6 were found to be significantly more elevated and those of GM-CSF lower in patients with infertility due to immunological causes as compared to those with tubal infertility. The former had also decreased intrafollicular E2 levels and increased progesterone concentrations. No difference was seen in the follicular fluid levels of testosterone and androstenedione. In conclusion, several macrophage-derived cytokines are present in the follicular fluids of infertile women. Patients with infertility due to immunological causes had higher levels of IL-6 and lower concentrations of GM-CSF as compared to patient with a tubal factor of infertility. We speculate that this abnormal cytokine profile may contribute to the altered intrafollicular steroid milieu.

The neuroactive steroid allopregnanolone suppresses hypothalamic gonadotropin-releasing hormone release through a mechanism mediated by the gamma-aminobutyric acidA receptor.

The central nervous system (CNS) is able to synthesize and/or metabolize steroid hormones. These neuroactive steroids are capable of modulating several brain functions and, among these, they seem to regulate the hypothalamic-pituitary-gonadal (HPG) axis. Indeed, recent observations have shown that 5 alpha-pregnane-3 alpha-ol-20-one (allopregnanolone), one of the most abundant naturally occurring neuroactive steroids, suppresses ovulation and sexual behaviour when administered within the CNS. The present study was undertaken to evaluate the effects of allopregnanolone and its inactive stereoisomer, 5 alpha-pregnane-3 beta-ol-20-one, upon the release of gonadotropin-releasing hormone (GnRH) from individually-incubated hemihypothalami. Allopregnanolone suppressed GnRH release in a concentration-dependent manner with maximal activity in the nanomolar range, a range at which this neurosteroid is capable of playing a biological action. The specificity of allopregnanolone suppression of GnRH release was provided by the lack of effect of its known inactive stereoisomer. To evaluate the involvement of gamma-aminobutyric acidA (GABAA) receptor, we examined the effects of two neurosteroids with GABA-antagonistic properties, pregnanolone sulfate (PREG-S) and dehydroepiandrosterone sulfate (DHEAS), and of bicuculline, a selective antagonist of the GABA binding site on the GABAA receptor, on allopregnanolone (10 nM)-suppressed GnRH release. Both PREG-S and bicuculline overcame the inhibitory effects of allopregnanolone on GnRH release, whereas DHEAS did not. To substantiate the involvement of the GABAA receptor further, we tested the effects of muscimol, a selective agonist for this receptor, which suppressed GnRH release. In conclusion, allopregnanolone suppressed hypothalamic GnRH release in vitro and this effect appeared to be mediated by an interaction with the GABAA receptor. We speculate that the inhibitory effect of allopregnanolone on the HPG axis may also be caused by its ability to suppress hypothalamic GnRH release.

Endothelin (ET)-1 and ET-3 inhibit estrogen and cAMP production by rat granulosa cells in vitro.

Endothelin (ET)-1 and ET-3, two peptides with a potent vasoconstrictive property, produce a variety of biological effects in different tissues by acting through two different receptors, the ET-1 selective ET(A) receptor and the non-selective ETB receptor. An increasing body of literature suggests that ET-1 acts as a paracrine/autocrine regulator of ovarian function. Indeed, ETB receptors have been identified in rat granulosa cells and ET-1 is a potent inhibitor of progesterone production. In contrast, inconsistent data have been reported about the role of ET-1 on estrogen production and the effects of ET-3 are not known. Therefore, the present study was undertaken to evaluate the effects of ET-1 and ET-3 on estrogen and cAMP production, and the receptor type involved. Given that prostanoids modulate ovarian steroidogenesis and that many actions of ETs are mediated by these compounds, we also evaluated whether the effects of ETs on estrogen and cAMP production might be prostanoid-mediated. ET-1, ET-3, and safarotoxin-S6c (SFX-S6c), a selective ETB receptor agonist, inhibited basal estrogen production by granulosa cells obtained from immature, estrogen-primed female rats, in a concentration-dependent manner. All three peptides were also capable of inhibiting the production of estrogen stimulated by a half-maximal (1 mIU/ml) and a maximally stimulatory (3 mIU/ml) concentration of FSH, ET-1 and ET-3 dose-dependently suppressed basal and FSH (1 mIU/ml)-stimulated cAMP production. ET-3 and SFX-S6c were significantly more potent than ET-1 in suppressing estrogen production, suggesting that this effect was not mediated by the ET(A) receptor. Indeed, BQ-123, a selective ET(A) receptor antagonist, did not influence the inhibitory effects of ET-1 and ET-3 on basal and FSH-stimulated estrogen release. To determine a possible involvement of prostanoids, we evaluated the effects of maximally effective concentrations of ET-1 and ET-3 on estrogen and cAMP production in the presence of indomethacin, a prostanoid synthesis inhibitor. This compound did not have any effect on the suppressive effects of ETs on basal or FSH (1 mIU/ml)-stimulated estrogen or cAMP production. In conclusion, ET-1 and ET-3 were able to inhibit estrogen and cAMP production by rat granulosa cells, indicating that the inhibitory effects of ETs on ovarian steroidogenesis are not limited to progesterone biosynthesis. This effect does not appear to be mediated by prostanoids or by the classical ET(A) and ETB receptors, at least under these experimental conditions.

Correlation between intracellular cAMP content, kinematic parameters and hyperactivation of human spermatozoa after incubation with pentoxifylline.

Various compounds have been used in the attempt to improve sperm motility, including pentoxifylline (PF), a methylxanthine derivative. It has been postulated that PF, being a phosphodiesterase inhibitor, increases sperm kinematic parameters and the number of spermatozoa exhibiting hyperactivated motility by raising the intracellular content of cAMP, a molecule involved in the generation of sperm energy. However, it has not been clarified whether the biological effects of PF on sperm motility correlate with its ability to increase intracellular cAMP levels. To examine this relationship, the kinematic parameters, hyperactivation, and intracellular cAMP content were evaluated in motile spermatozoa, obtained by discontinuous Percoll gradient and swim-up from 21 normozoospermic semen samples, incubated without and with PF for 0, 1, 2, and 4 h. PF increased beat cross frequency after 1 and 2 h of incubation, curvilinear velocity and lateral head displacement (ALH) after 4 h, and hyperactivation after 1, 2, and 4 h, and decreased linearity (LIN) after 1 h of incubation. The intracellular cAMP content of spermatozoa incubated with PF increased at all time-points examined. Both intracellular cAMP content and increase in hyperactivation in response to PF decreased with the length of incubation. In the absence of PF, cAMP content was unchanged and was correlated significantly only with ALH and the percentage of spermatozoa with hyperactivated motility. Following incubation with PF, cAMP content correlated with hyperactivation and all sperm kinematic parameters, with the exception of LIN and straightness. These findings suggest that the beneficial effects of PF on sperm kinematic parameters and hyperactivation are related to its ability to increase intracellular cAMP content.

Activin-A stimulates hypothalamic gonadotropin-releasing hormone release by the explanted male rat hypothalamus: interaction with inhibin and androgens.

The presence of activins in those hypothalamic regions containing gonadotropin-releasing hormone (GnRH)-secreting neurons suggests that these peptides may regulate the reproductive function modulating not only pituitary FSH release and biosynthesis, but also hypothalamic GnRH release. The purpose of this study was to evaluate the effects of activin-A, a homodimer of inhibin beta A subunit, on hypothalamic GnRH release in vitro and, because of their well known antithetical effects, to evaluate its interaction with inhibin. In addition, since androgens modulate the release of GnRH from male rat hypothalami, we thought it of interest to study the possible interplay between these steroids and activin on GnRH release. To accomplish this, we employed a hypothalamic organ culture system which enabled us to evaluate GnRH release from individually incubated hemi-hypothalami explanted from male rats. Activin-A stimulated GnRH release in a biphasic manner. The maximal effect was reached at a concentration of 10 ng/ml which increased GnRH output by about 75%. Inhibin abolished the stimulatory effect of a maximally effective concentration of activin-A in a dose-dependent manner, whereas alone it had no effect on GnRH output. As previously shown, testosterone (1 nmol/l) and dihydrotestosterone (DHT, 0.1 nmol/l) suppressed basal GnRH release, but only testosterone was able to inhibit the release of GnRH stimulated by activin-A. Since DHT is a non-aromatizable androgen, we evaluated whether the inhibitory effect of testosterone was due to its in vitro conversion into 17 beta-estradiol. The addition of 4-hydroxyandrostenedione, a steroidal aromatase inhibitor, did not influence the suppressive effect of testosterone on GnRH release stimulated by activin-A. In conclusion, activin-A stimulated hypothalamic GnRH release in vitro and this effect was abolished by inhibin and was blunted by testosterone. These findings suggest that activins may participate in the regulation of the hypothalamic-pituitary-gonadal axis by modulating GnRH release. The ability of testosterone to suppress the release of GnRH stimulated by activin-A indicates that this steroid has a potent negative feedback influence on GnRH release.

Identification, characterization, and biological activity of endothelin receptors in human ovary.

We previously reported the expression of endothelin-1 (ET-1) in granulosa cells (GCs) of the human ovary and the presence of ET-1-like immunoreactivity in human follicular fluid obtained from women in an in vitro fertilization program. In follicular fluid, but not in plasma, the levels of ET-1-like immunoreactivity were higher in gonadotropin-stimulated vs. spontaneous cycles, suggesting hormonal regulation of follicular ET-1. To identify and characterize ET receptors in human ovary, we performed autoradiography, radioligand binding, and functional studies. Mathematical analysis of families of self- and cross-competition curves among [125I]ET-1, [125I]ET-3, and selective analogs indicates that human ovary expresses both subtypes of ET receptors, i.e. ETA and ETB receptors. However, the concentration of the ETB site was 100-fold lower than that of the ETA one. By using [125I]ET-1, we demonstrated that the density of binding sites in human ovary is not affected by the hormonal milieu (similar concentrations in normal cycling, postmenopausal, and combined oral contraceptive-treated women). In situ binding studies indicate that the majority of ETA and ETB receptors are expressed in the blood vessels of the ovary. In particular, ETA receptors are abundant in the ovulatory follicles and localized in the theca interna, in close proximity to the granulosa layer. Few GCs of the ovulatory follicle were specifically labeled. Conversely, in the rat ovary, used as a control, ETB receptors were predominantly expressed and localized in GCs. Accordingly, ETB receptors negatively regulated estrogen accumulation in rat GCs. In human granulosa-luteal cells, neither ET-1 (unselective ligand) nor ET-3 or sarafotoxin 6c (ETB ligands) affected estrogen or progesterone secretion. ET-1 was 2.5-fold more potent than noradrenaline in eliciting contraction of ovarian artery, acting through the ETA receptor. Our results indicate that in human ovary, at variance with rat ovary, the endothelin system is primarily involved in the regulation of ovarian blood flow and not steroidogenesis.

Interaction between prolactin and catecholamines on hypothalamic GnRH release in vitro.

Brain catecholamines have been implicated in the regulation of gonadotrophin release. It has been recently reported that noradrenaline (NA), applied within the hypothalamic paraventricular nucleus, suppresses the pulsatile release of LH in the rat through a corticotrophin-releasing hormone (CRH)-dependent mechanism. Prolactin (PRL) is also able to suppress hypothalamic GnRH release following activation of the CRH-releasing neurone. Given that PRL stimulates the release of NA from hypothalamic explants and that NA stimulates the release of hypothalamic CRH, we hypothesized that this neurotransmitter may be involved in the intrahypothalamic neuroendocrine circuit mediating the inhibitory effects of PRL on GnRH release. To test this hypothesis, we evaluated the effects of PRL on GnRH release in the presence of alpha- or beta-adrenergic receptor antagonists using a static hypothalamic organ culture system which enabled us to evaluate immunoreactive GnRH (iGnRH) release from individually incubated, longitudinally halved hypothalami. As previously shown, PRL at a concentration of 100 nM inhibited basal iGnRH release by about 35%. Phentolamine, a non-selective alpha-adrenergic receptor antagonist, prazosin, an alpha 1-receptor antagonist, and yohimbine, an alpha 2-receptor antagonist, overcame the inhibitory effect of PRL on iGnRH release in a concentration-dependent fashion. In contrast, propranolol, a non-selective beta-adrenergic receptor antagonist, atenolol, a beta 1-receptor antagonist, and ICI-118,551, a beta 2-receptor antagonist, had no effect. None of these compounds had any effect on basal iGnRH release. These findings suggested that an alpha-adrenergic mechanism is involved in the suppressive effects of PRL on GnRH release. Since the activation of alpha-adrenergic receptors increases hypothalamic CRH release, we evaluated whether PRL stimulates CRH release via an alpha-adrenergic mechanism. PRL stimulated basal CRH release by about twofold and this effect was inhibited by phentolamine in a concentration-dependent fashion. In conclusion, alpha-, but not beta-, adrenergic receptors mediate the inhibitory effects of PRL on GnRH release in vitro. We speculate that, at least under these experimental conditions, PRL inhibits GnRH release through an alpha-adrenergic mechanism which activates the CRH-secreting neurone.

Effects of corticotropin-releasing hormone on ovarian estrogen production in vitro.

It is a common clinical observation that stress is accompanied by dysfunction of the hypothalamic-pituitary-ovarian axis, and there is mounting experimental evidence that CRH, the principal regulator of ACTH release and the central coordinator of the stress response, is able to suppress gonadal function by inhibiting hypothalamic GnRH release. Recently, it has been shown that immunoreactive CRH, CRH messenger RNA, and CRH receptors are also present in the ovary. This prompted us to examine the role of CRH on ovarian function. To accomplish this, we studied the effects of this neuropeptide on estrogen production and cAMP intracellular content from rat granulosa and human granulosa-luteal cells. We also evaluated the activity of the enzyme aromatase by measuring the production of tritiated water from homogenates of cultured rat granulosa cells. CRH inhibited FSH-stimulated estrogen production from rat granulosa cells in a dose-dependent fashion. The maximal effect was achieved at a concentration of 10(-8) M, which suppressed estrogen production by about 30%. Low concentrations of CRH (10(-10) M), incapable of modulating maximal estrogen production in response to FSH, provoked a right-ward shift of the estrogen dose-response curve to FSH. CRH (10(-8) M) suppressed the production of tritiated water (equivalent to estrogen production) from homogenates of rat granulosa cells incubated with a half-maximal concentration of FSH. Basal estrogen production by human granulosa-luteal cells was also inhibited by CRH at a concentration of 10(-10) M. The maximal effect was achieved with a concentration of 10(-8) M, which lowered estrogen production by 25%. The CRH receptor antagonist alpha-helical CRH-(9-41) antagonized the inhibitory effect of CRH on estrogen production from rat granulosa and human granulosa-luteal cells, whereas alone it had no effect. CRH did not have any effect on the intracellular cAMP content of rat granulosa and human granulosa-luteal cells. In conclusion, these results suggest that CRH is able to suppress estrogen production from rat and human granulosa cells in vitro. This effect seems to be linked to inhibition of aromatase activity in the rat and is independent of cAMP generation. We speculate that CRH may also interfere with hypothalamic-pituitary-gonadal axis function by acting directly at the ovarian level.