The evolution of colour pattern complexity: selection for conspicuousness favours contrasting within-body colour combinations in lizards.

Many animals display complex colour patterns that comprise several adjacent, often contrasting colour patches. Combining patches of complementary colours increases the overall conspicuousness of the complex pattern, enhancing signal detection. Therefore, selection for conspicuousness may act not only on the design of single colour patches, but also on their combination. Contrasting long- and short-wavelength colour patches are located on the ventral and lateral surfaces of many lacertid lizards. As the combination of long- and short-wavelength-based colours generates local chromatic contrast, we hypothesized that selection may favour the co-occurrence of lateral and ventral contrasting patches, resulting in complex colour patterns that maximize the overall conspicuousness of the signal. To test this hypothesis, we performed a comparative phylogenetic study using a categorical colour classification based on spectral data and descriptive information on lacertid coloration collected from the literature. Our results demonstrate that conspicuous ventral (long-wavelength-based) and lateral (short-wavelength-based) colour patches co-occur throughout the lacertid phylogeny more often than expected by chance, especially in the subfamily Lacertini. These results suggest that selection promotes the evolution of the complex pattern rather than the acquisition of a single conspicuous colour patch, possibly due to the increased conspicuousness caused by the combination of colours with contrasting spectral properties.

Using visual modelling to study the evolution of lizard coloration: sexual selection drives the evolution of sexual dichromatism in lacertids.

Sexual selection has been invoked as a major force in the evolution of secondary sexual traits, including sexually dimorphic colourations. For example, previous studies have shown that display complexity and elaborate ornamentation in lizards are associated with variables that reflect the intensity of intrasexual selection. However, these studies have relied on techniques of colour analysis based on human--rather than lizard--visual perception. Here, we use reflectance spectrophotometry and visual modelling to quantify sexual dichromatism considering the overall colour patterns of lacertids, a lizard clade in which visual signalling has traditionally been underrated. These objective methods of colour analysis reveal a large, previously unreported, degree of sexual dichromatism in lacertids. Using a comparative phylogenetic approach, we further demonstrate that sexual dichromatism is positively associated with body size dimorphism (an index of intrasexual selection), suggesting that conspicuous coloration in male lacertids has evolved to improve opponent assessment under conditions of intense male-male competition. Our findings provide the first evidence for the covariation of sexual dichromatism and sexual size dimorphism in lacertids and suggest that the prevalent role of intrasexual selection in the evolution of ornamental coloration is not restricted to the iguanian lineage, but rather may be a general trend common to many diurnal lizards.

Evidence for a role of the alternatively spliced ED-I sequence of fibronectin during ovarian follicular development.

This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor messenger RNA, play specific roles during development of the ovarian follicle. In particular, we were interested in determining the effect of the ED-I (also termed ED-A) type III repeat, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I+ FN, whereas total FN levels remained relatively constant. ED-I+ FN levels were higher in small follicles, corresponding to the phase of granulosa cell proliferation. The hypothesis of a physiological role for ED-I+ FN was further supported by the finding of a regulation of the alternative splicing of FN in primary cultures of bovine granulosa cells by factors known to control ovarian follicular development. cAMP produced a 10-fold decrease in the relative proportion of the ED-I region. In contrast, transforming growth factor-beta elicited a 2-fold stimulation of overall FN synthesis and a 4-fold increase in the synthesis of ED-I containing FN. This effect was evident at the protein (Western blots) and messenger RNA (Northern blots) levels. Although a negative correlation (P < 0.001) was detected between ED-I+ FN and estradiol levels in follicular fluid, this steroid was unable to modulate in vitro the alternative splicing of FN. A possible mitogenic effect of ED-I+ FN was suggested by the observation that a recombinant peptide corresponding to the ED-I domain stimulated DNA synthesis in a bovine granulosa cell line (BGC-1), whereas a peptide corresponding to the flanking type III sequences had no effect. The hypothesis of ED-I+ FN as a growth regulatory factor was further strengthened by the fact that depletion of FN from BGC-1-conditioned medium, which contained ED-I+ FN, abrogated its mitogenic activity, whereas plasma FN was without effect. We propose that changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.

Dimeric inhibin A and B production are differentially regulated by hormones and local factors in rat granulosa cells.

In the present study, we have examined the role of hormones and growth factors in regulating dimeric inhibin production in immature rat granulosa cells. Purified granulosa cells from estrogen-primed immature rats were cultured under defined conditions. Inhibins A and B in the culture media were measured using a two-site enzyme-linked immunosorbent assay specific for each dimer. Under basal conditions, granulosa cells produced 14-fold more inhibin A than inhibin B (inhibin A, 2.0; inhibin B, 0.14 ng/ml, measured against human standards; average A/B apparent ratio, 14). Addition of increasing doses of FSH elicited dose-dependent increases in both inhibins, the effects being more pronounced on inhibin A than on inhibin B (9.4- and 4.1-fold increases, respectively; average A/B ratio, 34). Estradiol, when added alone, stimulated inhibin A production 3- to 6-fold, whereas minor changes were observed in inhibin B production. Insulin-like growth factor-I produced a similar stimulation of both inhibins (3-fold stimulation over control). This growth factor, however, induced a marked dissociation in the sensitivity of inhibins A and B to FSH stimulation, with maximal stimulation of inhibin B observed at comparatively lower concentrations of the gonadotropin. Transforming growth factor-beta (TGF-beta, 5 ng/ml) had a more marked stimulatory effect on inhibin B than on inhibin A production (7- to 14-fold vs. 2- to 5-fold for inhibin B and A, respectively). A more pronounced differential stimulation of inhibin B was also exerted by another member of the TGF-beta superfamily, activin A (A/B ratio, 0.66). This preferential stimulation of inhibin B by TGF-beta and activin A was amplified in the presence of FSH. Coculture of rat granulosa cells with freshly isolated bovine oocytes was also associated with a marked stimulation of inhibin B production (100-fold increase) and a comparatively lower stimulation of inhibin A (10-fold increase; A/B ratio, 1). The discrepancy between the proportion of inhibin dimers in serum (A/B ratio, 0.13) and those produced by untreated granulosa cells may suggest that intraovarian factors, such as TGF-beta, activin A, or oocyte-derived factor(s), are responsible for the shift of the ratio toward the predominance of inhibin B.

Growth promoting activity of oocytes on granulosa cells is decreased upon meiotic maturation.

An increasing body of evidence indicates that the oocyte plays an active role in the control of ovarian follicle development in mammals. In the present study, we have examined the role of oocytes in regulating granulosa cell proliferation. Rat and bovine oocytes cocultured with rat granulosa cells stimulated granulosa cell DNA synthesis and DNA content in the cultures. FSH or cAMP further amplified this effect. Poor-quality oocytes showed a marked decrease in their stimulatory effect. Stimulation of DNA synthesis by bovine oocytes seems to be cell-type specific, since Swiss 3T3 fibroblasts and CCL-64 mink lung epithelial cells were not responsive, while primary cultures of rat and bovine granulosa cells and the bovine granulosa cell line BGC-1 showed significant responses. Oocyte-conditioned medium produced only a slight stimulation of rat granulosa cell DNA synthesis. However, the effect of oocyte coculture was dependent on the total incubation volume, suggesting that the growth promoting activity was mediated by a soluble factor. The stimulation elicited by bovine oocytes was evident even in the presence of maximally effective doses of transforming growth factor-beta or tumor necrosis factor-alpha, indicating that neither of these growth factors was responsible for this effect. In vitro maturation of bovine oocytes was associated with a marked decrease in the stimulatory activity. This decrease was partially prevented when maturation was blocked by addition of cycloheximide. Comparison of the developmental pattern of the secretion of the growth promoting activity with that of the cumulus expansion-enabling factor indicated that both activities can be dissociated. Our data suggest the existence of a very labile factor produced by the oocyte before completion of the first meiotic division that promotes granulosa cell proliferation.

Inhibitory effect of gonadotrophin-releasing hormone (GnRH) on rat granulosa cell deoxyribonucleic acid synthesis.

Gonadotropin-releasing hormone (GnRH) has been found to be expressed within the ovary and to modulate cell differentiation in ovarian cells. In the present study we have analyzed the influence of GnRH on DNA synthesis in rat granulosa cells. Cells were obtained from immature DES-treated rats and cultured in defined medium (DMEM:F12) containing combinations of FSH, estradiol, and transforming growth factor-beta (TGF-beta), both in the presence and absence of GnRH. A GnRH analog, Leuprolide (GnRHa), caused a dose-dependent inhibition of 3H-thymidine incorporation in cells cultured in the presence of FSH (20 ng/ml) and TGF beta (2.5 ng/ml), at concentrations as low as 5 x 10(-11) M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED50 = 1.58 +/- 0.22 x 10(-10) M) and native GnRH (ED50 = 1.4 +/- 0.3 x 10(-6) M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide 10(-8) M could prevent the inhibition elicited by 10(-7) M of Leuprolide. These results suggest that GnRH may play a role in the regulation of rat granulosa cell proliferation during follicular development.

[Role of different forms of fibronectin in in vitro bovine follicular development]. / Funcion de distintas formas de fibronectina en el desarrollo de foliculos bovinos in vitro.

This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor mRNA, play specific roles during follicular development. In particular, we analyzed the presence of the ED-I region, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I + FN whereas total FN levels remained relatively constant. A negative correlation (P < 0.001) was detected between ED-I + FN and estradiol levels. This steroid was without effect on the alternative splicing of FN in primary cultures of bovine granulosa cells. However, cAMP produced a marked decrease in the incorporation of the ED-I region. In contrast, transforming growth factor beta (TGF-beta) elicited both a stimulation on overall FN synthesis and an increase in the inclusion of ED-I. This effect was evident at the protein level (Western blots) and also in the mRNAs (Northern blots). A peptide corresponding to the ED-I region stimulated DNA synthesis in a bovine granulosa cell line (BGC-1) whereas the peptide corresponding to the flanking sequences was without effect. Data presented herein suggest a novel form of regulation by which changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.

Immunoglobulins from human follicular fluid induce the acrosome reaction in human sperm.

The ability of human follicular fluid (hFF), retrieved from women undergoing IVF to induce the acrosome reaction (AR) in human sperm has been documented by several laboratories. However, the nature of the active factors in the hFF and the physiological meaning of the AR induction are highly controversial. We performed a three step purification scheme for hFF and all the fractions were screened for the AR-inducing activity. AR activity was associated with a protein fraction of M(r) > 180 kD that on further analysis under PAGE was found to be composed by subunits of apparent M(r) 50,000 and 29,000. The N-terminal sequences of these bands showed a 100% homology with the heavy and light chains of human IgG. A polyclonal antibody raised against the purified protein and anti-human IgG were both able to suppress the acrosome reaction-inducing activity of crude hFF. However, neither normal human serum nor a purified preparation of human IgG were able to mimic the AR-inducing activity of hFF. We concluded that the AR-inducing activity of hFF is, at least in part, due to the presence of antisperm antibodies.