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Gene duplication is considered an important evolutionary mechanism leading to new gene functions. According to the classical model, one gene copy arising from gene duplication retains the ancestral function, whilst the other becomes subject to directional selection for some novel functions. Hence, according to this model, long-term persistence of two paralogous genes is possible only with the acquisition of functional innovation. In the absence of neofunctionalization, one of the duplicate genes may be lost following accumulation of deleterious mutations, ultimately leading to the loss of function. Recently, new mechanisms have been proposed according to which both paralogs are maintained without apparent neofunctionalization. In this paper we describe the molecular evolution of the aspartic proteinase gene family, with particular regard for the nothepsin gene, a sex- and tissue-specific form of aspartic proteinase active in fish. The finding of nothepsin in a reptile is indicative of the presence of this gene in organisms other than fish. However, the failure to find any nothepsin-like gene in avian, murine and human genome suggests that the gene has been lost in certain lineages during evolution. At variance with piscine nothepsin expressed exclusively in female liver under the estrogens action, the reptilian counterpart lacks both tissue and sex specificity, as it is constitutively expressed in different tissues of male and female specimens. The expression of the nothepsin gene in fish and lizard is accompanied by the expression of a paralogous gene encoding for cathepsin D. Functional divergence analysis indicates that cathepsin D accumulated amino acid substitutions, whereas nothepsin retained most of the ancestral functions. Phylogenetic analysis shows a preponderance of replacement substitutions compared to silent substitutions in the branch leading to the cathepsin D clade, whilst nothepsin evolves under negative selection. To explain the loss of the nothepsin gene in certain lineages, we propose a model that takes into account the complementary degenerative mutations occurring in regulatory elements of the promoter regions of the two genes. According to this model, gene loss occurs whenever the two genes acquire the same expression pattern. The coexistence of cathepsin D and nothepsin is explained in terms of metabolic cooperation of the two enzymes.

The present paper reports the full nucleotide sequence of a cloned cDNA prepared from RNA of lizard ovaries. The open reading frame consists of 2019 nucleotides, which encodes a protein of 673 amino acids belonging to the G protein-coupled receptor superfamily with a large extracellular N-terminal domain involved in hormone recognition. The transmembrane domain ends with a short intracytoplasmic COOH-terminal domain involved in effector activation. Phylogenetic analysis showed that the lizard receptor belongs to the family of follicle-stimulating hormone (FSH) receptors. The hydrophobicity profile is similar to that observed for mammalian and avian FSH receptors. Northern blot analysis of total RNA revealed that the FSH receptor is expressed at high levels in the ovary. In situ hybridization experiments demonstrate that FSH receptor mRNA is specifically localized within the small cells of the follicular epithelium surrounding the oocyte.

The study of environmental factors affecting vertebrate reproduction has long interested both developmental and evolutionary biologists. Although photoperiod has been considered to be an important environmental parameter for vertebrates such as birds, temperature is probably a primary external factor responsible for reproductive cyclicity in reptiles. In spite of the progress made in the understanding of reptilian reproductive strategies and adaptations, much remains to be learned about the interplay between endocrine physiological factors, such as hormones, and environmental parameters. In this report, we have examined the effects of in vivo administered FSH on oocyte recruitment during the most significant periods of the reproductive cycle of the lizard, Podarcis sicula. The results show that when FSH is administered in proximity to the reproductive period, it stimulates oocyte growth and ovulation; when the hormone is administered at the beginning of the winter stasis it affects ovarian activity without inducing ovulation. Ovarian adenylate cyclase activity is moderately sensitive to in vitro FSH stimulation during the pre- and post-reproductive periods. The sensitivity to hormone stimulation increases significantly during the reproductive period and winter stasis. We have also tested the hypothesis that environmental temperature affects the responsiveness of ovarian adenylate cyclase to FSH stimulation. For such a purpose, we exposed animals to 28 degrees C or 4 degrees C in different periods of the ovarian cycle. The results show that, whenever the temperature applied mimics the thermal regime of the coming season, adenylate cyclase sensitivity to FSH shifts towards levels that anticipate the natural responsiveness.

During vitellogenesis, the oocytes of oviparous species accumulate in the cytoplasm a large amount of proteic nutrients synthetized in the liver. Once incorporated into the oocytes, these nutrients, especially represented by vitellogenin (VTG) and very low-density lipoprotein (VLDL), are cleaved into a characteristic set of polypeptides forming yolk platelets. We have studied the molecular mechanisms involved in yolk formation in a reptilian species Podarcis sicula, a lizard characterized by a seasonal reproductive cycle. Our results demonstrate the existence in the lizard ovary of an aspartic proteinase having a maximal activity at acidic pH and a molecular mass of 40 kDa. The full-length aspartic proteinase cDNA produced from total RNA by RT-PCR is 1,442 base pairs long and encodes a protein of 403 amino acids. A comparison of the proteic sequence with aspartic proteinases from various sources demonstrates that the lizard enzyme is a cathepsin D. Lizard ovarian cathepsin D activity is maximal in June, in coincidence with vitellogenesis and ovulation, and is especially abundant in vitellogenic follicles and in eggs. Ovarian cathepsin D activity can be enhanced during the resting period by treatment with FSH in vivo. Northern blot analysis shows that cathepsin D mRNA is exceedingly abundant during the reproductive period, and accumulates preferentially in previtellogenic oocytes.

Gonadotropins (FSH and LH) affect several mammalian gonadal functions. In particular, FSH stimulates oogonial proliferation and oocyte growth, while LH regulates ovulation and progesterone secretion. In lacertilian reptiles, gonadal function is also regulated by pituitary gonadotropins, but which hormone controls ovarian activities and the mechanisms of action are unknown. The present study aimed to clarify mechanisms of action of pituitary gonadotropins on the ovary of Podarcis sicula (Lacertilia). The data demonstrate that mammalian gonadotropins FSH and LH produce a threefold stimulation of adenylate cyclase activity in follicular membranes, while hCG and TSH are less effective, causing a twofold increase in adenylate cyclase activity. Neurotransmitters such as dopamine, serotonin, and catecholamines have no effect on enzyme activity. The action of mammalian FSH and LH on the ovary mimics the effect of homologous hormones: in lizard ovaries incubated in vitro in the presence of isolated homologous pituitary glands, the intracellular cAMP level increased by 50% with respect to control ovaries. Mammalian gonadotropins appear homologous to lizard gonadotropin(s): Southern blot analyses show that the lizard genome contains nucleotide sequences homologous to those encoding for mammalian beta FSH and beta LH. Both homologous and heterologous desensitization of adenylate cyclase activity occurs in the lizard ovary. In fact, responsiveness of adenylate cyclase to gonadotropin stimulation is abolished in animals 2 hr after in vivo treatment with FSH. Sensitivity to gonadotropin stimulation is restored 2 weeks after the beginning of the in vivo treatment. Desensitization was also observed in ovaries incubated in vitro with mammalian FSH or with isolated pituitary glands.