Progesterone in vitro increases growth, motility and progesterone receptor expression in third stage larvae of Toxocara canis.

The in vitro effect of progesterone in T. canis larvae on their enlargement and motility were evaluated, together to the possible presence of progesterone receptors (PRs). T. canis larvae were cultured in RPMI-1640 with different concentrations of progesterone (0, 20, 40, 80, 400 and 800 ng/mL). Enlargement and increases in motility were dependent on the concentration only from 0 to 80 ng/mL (p < 0.05). The mean percentage of PR + cells in newly obtained larvae as measured by flow cytometry was 8.16 ± 0.4. The number of PR + cells increased depending on concentration from 0 to 80 ng/mL (p < 0.001). Cells obtained from larvae stimulated at any of the studied hormone concentrations showed greater mean fluorescence intensity when compared to non-stimulated cells. Additionally, the expression and location of PR + cells were determined in the larvae. The sequence of an amplicon (420-bp) obtained by PCR from T. canis larvae showed 100% homology with a gene fragment that codes for the PR of the dog. PR + cells were immunolocated using confocal microscopy in the intestinal region of the larvae that had been recently obtained. The results of this study show that T. canis larvae can recognize and respond to the presence of progesterone through a molecule possibly able to bind it. Since we previously observed a similar response to prolactin, we suggest that both hormones could participate sequentially in the reactivation of T. canis larvae in pregnant bitches.

Progesterone inhibits the in vitro L3/L4 molting process in Haemonchus contortus.

We evaluated the direct effects of progesterone on the morphology, maturation and behavior of Haemonchus contortus larvae in vitro. The presence and location of possible progesterone receptors in these larvae were also determined. The addition of 8ng/mL of progesterone to larval cultures over 10days reduced larval enlargement, while the addition of 160ng/mL of the hormone increased the enlargement. Up to 62% and 65% of the H. contortus larvae molted from third-stage larvae (L3) to fourth-stage larvae (L4) when cultured in RPMI-1640 media without hormone for 5 and 10days, respectively. The addition of different progesterone concentrations (1, 8, 16, 80 and 160ng/mL) to the larval cultures significantly inhibited the molting process within the same periods. The addition of 8ng/mL or higher progesterone concentrations to the cultures significantly increased larval motility (p<0.05) compared with unstimulated larvae. Flow cytometry showed the expression of progesterone receptors (P4-R) in 15% of the cells from newly isolated H. contortus larvae. When the larvae were cultured for 5days in the presence of the hormone, the percentage of P4-R+ cells remained the same. In contrast, unstimulated larvae showed a significant reduction in the number of P4-R+ cells. Using confocal microscopy, a greater concentration of P4-Rs was immunolocated in the anterior portion of the alimentary tract of the larvae, suggesting that the cells in this region are targeted by the hormone. The results of the present study show that H. contortus larvae have possible P4-Rs and respond to this hormone by inhibiting their molting process, thereby suggesting the participation of progesterone in the larval arrest phenomenon.

The in vitro effect of prolactin on the growth, motility and expression of prolactin receptors in larvae of Toxocara canis.

The in vitro effect of prolactin (PRL) on the growth and motility of Toxocara canis larvae was assessed. Additionally, the expression and location of prolactin receptors (PRL-Rs) were determined in the larvae. Larvae of T. canis were incubated with different concentrations of PRL for different periods of time. The stimulated larvae accelerated their enlargement and increased their motility. The mean percentage of PRL-R+ cells in non-stimulated larvae, measured by flow cytometry was 7.3±0.3%. Compared with non-stimulated larvae, the mean fluorescence intensity (p<0.05) increased in larvae incubated with 40ng/mL of PRL for 10 days. A 465-bp length fragment was amplified from larvae gDNA by PCR. The sequence of this fragment showed 99% similarity with the gene fragment that codes for the PRL-R of the domestic dog. A high concentration of PRL-Rs was immune-located in the posterior region of the larval intestine; therefore, the intestinal cells in this region were most likely the targets for this hormone. Based on these results, PRL-Rs were identified in T. canis larvae, and the in vitro stimulation with PRL increased the number of these receptors, accelerated the growth and modified the activity of larvae. All of the above suggest that T. canis larvae are evolutionarily adapted to recognize the PRL of their definitive host and furthermore might explain the reactivation of tissue-arrested larvae during the gestation of bitches, which does not occur in gestating females of other species.

Observation of fresh Bos indicus embryos comparing stereoscopic and phase contrast microscopy.

Summary The precision of embryo evaluation using stereoscopic microscopy (SM) and inverted phase contrast microscopy (PCM) was compared in 20 Bos indicus cows superovulated at two different times of the year. In total, 118 embryos were collected and classified according to their developmental stage and quality by two independent evaluators using SM and inverted PCM. Cohen's kappa coefficient was used to determine concordance between SM and PCM observations. A good level of agreement (k = 0.616) was found for quality level, and a moderate one (k = 0.464) for developmental stage, particularly at the morula stage. Using the TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) technique, concordance level was deemed to be low with the SM (k = 0.169), and poor with the PCM (k = 0.217). Differences in concordance levels were also found between observations made at the two times of year, 78 embryos were evaluated in the rainy season when concordance level was good (k = 0.68), in contrast to the 40 embryos evaluated in the dry season when agreement was found to be poor (k = 0.24). In conclusion, inverted PCM was somewhat more effective for evaluating embryos, particularly at the morula stage. However, considering the high cost of an inverted PCM, the differences observed do not justify its purchase for routine embryo evaluation.

Assessment of the viability of embryos stored in liquid nitrogen produced commercially using culture medium as a complementary test for stereoscopic microscopy.

Summary The objective of the present study was to evaluate the viability of frozen embryos obtained from various private farmers in a culture medium for 4 h. Forty-seven embryos were used that had been previously graded as good and fair. These embryos were evaluated using stereoscopic microscopy by experienced clinicians prior to freezing. Embryos were divided in two groups: the non-cultured group, made up of six good quality embryos, and five fair; and the cultured group that consisted of 20 good quality embryos and 16 fair. Fifty-four per cent of the good quality embryos achieved a favourable development during culture whereas just 42% of embryos determined to be fair were observed to have adequate development. This evaluation was undertaken by serial photographs obtained at the onset of culture and 4 h later. This finding was corroborated by a more specific technique: terminal deoxynucleotide transferase dUTP nick end labelling-bromodeoxyuridine (TUNEL-BrdU). These results are indicative of the necessity of tight quality controls for commercially produced frozen embryos, as once thawed it is unlikely that clinicians will examine them to determine their physiological status prior to transfer.

Gonadal morphogenesis and sex differentiation in the viviparous fish Chapalichthys encaustus (Teleostei, Cyprinodontiformes, Goodeidae).

This study describes the structural and ultrastructural characteristics of gonadal sex differentiation and expression of Vasa, a germline marker, in different developmental stages of embryos and newborn fry of the barred splitfin Chapalichthys encaustus, a viviparous freshwater teleost endemic to Mexico. In stage 2 embryos, the gonadal crest was established; gonadal primordia were located on the coelomic epithelium, formed by scarce germ and somatic cells. At stage 3, the undifferentiated gonad appeared suspended from the mesentery of the developing swimbladder and contained a larger number of germ and somatic cells. At stages 4 and 5, the gonads had groups of meiotic and non-meiotic germ cells surrounded by somatic cells; meiosis was evident from the presence of synaptonemal complexes. These stages constituted a transition towards differentiation. At stage 6 and at birth, the gonad was morphologically differentiated into an ovary or a testis. Ovarian differentiation was revealed by the presence of follicles containing meiotic oocytes, and testicular differentiation by the development of testicular lobules containing spermatogonia in mitotic arrest, surrounded by Sertoli cells. Nuage, electron-dense material associated with mitochondria, was observed in germ cells at all gonadal stages. The Vasa protein was detected in all of the previously described stages within the germ-cell cytoplasm. This is the first report on morphological characteristics and expression of the Vasa gene during sexual differentiation in viviparous species of the Goodeidae family. Chapalichthys encaustus may serve as a model to study processes of sexual differentiation in viviparous fishes and teleosts.

Use of a culture medium (McCoy®), as a method for evaluating Bos indicus × Bos taurus embryos.

The aim of this study was to use culture medium (McCoy®) as a test to evaluate the classification of embryos after a primary grading using stereoscopic microscopy to further confirm whether embryos have been correctly scored by stereoscopic microscopy evaluating the level of apoptosis. Forty-six Bos indicus embryos were collected with a non-surgical method and evaluated with stereoscopic microscopy for scoring in three categories (good, fair and poor). Cell proliferation and apoptosis were assessed and compared between the control group (n = 14) at the onset of the experiment and in an experimental group (n = 32) after stored for 4 h in a culture medium. Embryos were processed using TUNEL and BrdU markers to determine the apoptosis and cell proliferation. Seventy-four per cent of good quality embryos developed favourably after the 4 h of culture; 60% of fair embryos maintained their evolution, while 100% of poor quality embryos presented degenerative changes from the beginning. No statistical differences were found between the experimental and the control groups in the count of positive BrdU and apoptotic nuclei. In poor quality embryos, a higher number of apoptotic cells were found relative to good and fair embryos, both in the experimental and control groups (P < 0.05). These results suggest that the culture medium may be used for a short time as a fast, practical and non-invasive alternative to further confirm whether embryos have been correctly scored by stereoscopic microscopy.

Expression of 3ß-HSD1 and P450 Aromatase enzymes during mouse gonad differentiation.

In sheep embryos, steroidogenic activity has been reported as taking place during the period of sexual differentiation. In the case of mouse embryos, the sporadic detection or absence of steroidogenic enzymes suggests that the ovary is inactive. The purpose of this work was to establish if mouse undifferentiated gonads express steroidogenic enzymes in a similar way as in sheep embryos. To know this, we analyzed the mRNA expression pattern of 3ß-Hsd1 and P450arom as well as protein expression pattern of 3ß-HSD1 and Testosterone in normal undifferentiated and differentiated gonads from both male and female mice embryo. Our data indicate that there is expression of 3ß-Hsd1 in XX gonads during gonad differentiation period. Nevertheless the Testosterone which would indicate steroidogenic activity is not produced. Besides, the absence of P450arom indicates that the production of Estradiol as observed in the ovaries of sheep does not occur. The detection of 3ß-Hsd1 in the early stages of ovarian development, as well as the absence of Testosterone suggests that XX gonads are not steroidogenic and that 3ß-Hsd1 enzyme may play a different role than in the steroidogenesis process.

Gonadectomy inhibits development of experimental amoebic liver abscess in hamsters through downregulation of the inflammatory immune response.

Incidence of amoebic liver abscess (ALA) in human males is considerably higher than in females, suggesting a role for sex hormones in this parasite infection. We describe here the effect of hamster gonadectomization on the development of ALA. After monitoring the decrease of oestradiol in females and testosterone in males to undetectable levels by ELISA and Radio Immuno Assay (RIA) in serum, hamsters were intraportally infected with Entamoeba histolytica trophozoites and killed 7 days later. ALA was absent in 50% of male and 15% of female gonadectomized (Gdx) hamsters, in comparison with 100% infection in non-Gdx controls. This protection against ALA in Gdx hamsters was concomitant to a comparatively scarce inflammatory infiltrate and necrosis surrounding clusters of trophozoites in the liver tissue, as well as to a lack of response of spleen cells to Con A, evaluated in proliferation assays. As tissue damage in ALA has been associated with a local inflammatory Th1 response, we determined the profile of response in hamsters by immunohistochemistry on liver sections. In contrast to strong Th1 responses in non-Gdx animals, Gdx females and males exhibited Th2 and Th3 profiles of cytokines, respectively, suggesting that protection against ALA following gonadectomization, could be related to downregulation of liver Th1 response during amoebic infection.

A system to evaluate the quality of frozen embryos through short-term culture.

The aim of the present study was to evaluate a culture system as a non-invasive approach intended for assessing the viability of recently thawed embryos prior to transfer. Embryos (n=51) were collected seven days after insemination out of 20 cows that had been treated to synchronize estrus and induce superovulation. Embryos were classified as good, fair, and poor and frozen. All embryos were cultured in McCoy medium. Morphology was monitored for a period of 24h to register the development stage every 30 min for the first 2h, and every hour thereafter. A sample of four embryos of each classification was separated at 4h, another four at 12h, and the remaining seven at 24h and the degree of apoptosis was determined for all the embryos using the TUNEL technique. Embryos of good and fair quality did not undergo major detrimental changes in development even after 7h of incubation, whereas poor quality embryos experienced changes as early as 2h after incubation. Good quality embryos invariably had fewer numbers of apoptotic cells than those of fair and poor quality suggesting that embryo culture can be a useful method to assess viability and to confirm the quality of thawed embryos previously stored in liquid nitrogen prior to transfer.

Evidence of damage in cryopreserved and fresh bovine embryos using the Tunel technique.

The objective of the present study was to evaluate the quality of bovine embryos cryopreserved in different years in Chiapas, Mexico. The embryos were obtained from a government institution (FIMEGEN) dedicated to promoting embryo transfer among dual-purpose cattle farmers. Forty-three embryos frozen in 1988, 1989, 2000 and 2002 were analysed with the Tunel technique to detect programmed cell death (apoptosis). Eleven fresh embryos were used as controls. Analysis of variance was used in embryos stored in the different years with averages tested using Tukey's test. Student's t-test was employed to compare fresh and frozen cells. Embryos with shorter storage time presented a lower number (p < 0.001) of Tunel-positive cells compared with embryos stored for longer time. On the contrary, when comparing the number of apoptotic cells between frozen and fresh embryos a higher number of positive cells (p < 0.05) were found in the former. The present results suggest that the cryopreservation per se caused damage that compromises the viability of the embryo. Another explanation for the lower pregnancy rate found in the tropics could be irreversible damage caused by poor storage technique in these large operations.

Cell aggregation precedes the onset of Sox9-expressing preSertoli cells in the genital ridge of mouse.

SOX9 is expressed at the onset of the genital ridge formation in both sexes. It is assumed that SRY, the testis determining gene, turns SOX9 on in male embryos because it is turned off in female embryos. Spatial expression of SRY follows a cranio-caudal pattern. Here, we asked if SOX9 is expressed in the same cell lineage and with a similar pattern as SRY. A correlative study between the structural changes in the genital ridge and the immunocytochemical localization of SOX9-positive cells was undertaken. We used a transgenic strain expressing the green fluorescent protein (GFP) that considerably enhanced the cell context where the first SOX9-positive cells appear. Although SOX9-positive cells are located among loose mesenchymal cells by stages of 8-14 tail somites (ts) in both sexes, they are absent in the thickening coelomic epithelium of females. At 15 ts the first SOX9-positive cells appear within the core of the condensed cells only in male genital ridges. At 17 ts, a gradient of SOX9-positive cells in males is apparent, closely following the cranio-caudal pattern of cell aggregation seen in genital ridges of both sexes. Hence, our results suggest that SOX9 is expressed only in loose mesenchymal cells in both sexes and that expression of SOX9 in males requires the prior aggregation of cells in the genital ridges. The correspondence of SOX9 and SRY pattern of expression supports that both genes are expressed in the preSertoli cell lineage in the core of the genital ridges.

Expression profiles of Dax1, Dmrt1, and Sox9 during temperature sex determination in gonads of the sea turtle Lepidochelys olivacea.

Sex determination is controlled either by genetic or environmental factors. In mammals Sry initiates determination but no homologue of this gene exists in non-mammalian species. Other genes of the mammalian sex-determining pathway have been identified in gonads of different vertebrates. Sox9, Dax1, and Dmrt1 are expressed at the onset of gonadal development in birds and reptiles. In the sea turtle Lepidochelys olivacea, a species with temperature sex determination (TSD), Sox9 is expressed in undifferentiated gonads at male- (MPT) or female-promoting temperatures (FPT). At MPT, Sox9 remains expressed in male gonads, but at FPT it is downregulated coinciding with the onset of the ovarian morphologic differentiation and female sex determination. At MPT however, male sex is determined early than at FPT in still undifferentiated gonads suggesting that other genes maintain Sox9 expression in testis. Here we used RT-PCR to study the expression profiles of Dax1, Dmrt1, and Sox9 in gonads of embryos of L. olivacea incubated at MPT or at FPT. The profiles were correlated with sex determination during and after the temperature-sensitive period (TSP). Dax1 maintained similar levels at both temperatures during the TSP. The Dax1 expression level increased significantly in ovaries compared to testes at stage 27, once they were morphologically distinct. The expression levels of Dmrt1 were higher at MPT than at FPT at all stages, in contrast with Sox9 levels which were similar at both temperatures at stages 23-25. Together, current results suggest that, whereas Dax1 is not involved in TSD in L. olivacea, upregulation of Dmrt1 and downregulation of Sox9 may play a role in male and female sex determination, respectively.

Onset of sex differentiation: dialog between genes and cells.

During the late 1940s, Alfred Jost demonstrated that mammalian sex differentiation begins in fetal testis, producing two factors necessary for the establishment of phenotypic males. Castrated embryos prior to testis differentiation led to phenotypic female differentiation. Jost proposed the existence of a testis-determining factor (TDF), elucidated in 1990 and named SRY for humans and Sry for mice. Thereafter, an increasing list of genes expressed in the genital ridges of mouse embryos at the onset of gonad differentiation has appeared. To date, it is clear that complete understanding of the mechanisms underlying gonadal sex differentiation in mammals requires identification of key cell lineages in which gonadal-specific genes are expressed. Here, a correlation between known gene expression and gonadal morphologic changes is attempted.

Timing of SOX9 downregulation and female sex determination in gonads of the sea turtle Lepidochelys olivacea.

The SRY-related gene SOX9 is involved in the differentiation of Sertoli cells in male gonads of vertebrates with different kinds of sex determination. In the olive ridley Lepidochelys olivacea, a species with temperature sex determination (TSD), the SOX9 protein is expressed at stages 21-24 in medullary cells in gonads of embryos incubated at both male-(MPT) or female-promoting temperatures (FPT). However, at FPT the expression of SOX9 protein decreases at stage 25 and disappears at stage 26, suggesting this as the critical period for SOX9 regulation by temperature. Here, we used reverse transcriptase polymerase chain reaction (RT-PCR) to detect SOX9 transcripts in gonads of embryos switched from MPT to FPT at stage 23 and sampled at days 6-14. Simultaneously, groups of embryos were switched back to MPT and gonadal sex was established. SOX9 transcripts were detected at days 6-12 of switching, when embryos reached stage 25 and were no longer detected at day 14, when the embryos were at stage 26. Embryos switched back to MPT at days 6 or 8 formed testes, whereas embryos switched at days 10 or 14 developed ovaries. Results suggest that at MPT the male sex-determining pathway that maintains SOX9 expression in male gonads is established at stage 24. In contrast, at FPT, the female sex-determining pathway involved in downregulation of SOX9 in female gonads occurs within two days at stage 25. J. Exp. Zool. 290:498-503, 2001.

Temperature regulates SOX9 expression in cultured gonads of Lepidochelys olivacea, a species with temperature sex determination.

Although sex determination starts in the gonads, this may not be the case for species with temperature sex determination (TSD). Since temperature affects the whole embryo, extragonadal thermosensitive cells may produce factors that induce gonadal sex determination as a secondary event. To establish if gonads of a species with TSD respond directly to temperature, pairs of gonads were cultured, one at female-promoting temperature (FPT) and the contralateral at male-promoting temperature (MPT). Histological and immunohistochemical detection of SOX9 revealed that the response to temperature of isolated gonads was similar to that of the gonads of whole embryos. While gonads cultured at MPT maintained SOX9 expression, it was downregulated in gonads at FPT. Downregulation of SOX9 took longer in gonads cultured at stage 23 than in gonads cultured at stage 24, suggesting that a developmental clock was already established at the onset of culture. To find out if sex commitment occurs in vitro, gonads were switched from FPT to MPT at different days. Results showed that the ovarian pathway was established after 4 days of culture. The present demonstration that gonads have an autonomous temperature detector that regulates SOX9 expression provides a useful starting point from which the molecular pathways underlying TSD can be elucidated.

Human tumor growth is inhibited by a vaccinia virus carrying the E2 gene of bovine papillomavirus.

BACKGROUND: Papillomavirus is the etiologic agent associated with cervical carcinoma. The papilloma E2 protein is able to regulate negatively the expression of E6 and E7 papilloma oncoproteins. Therefore, a new, highly attenuated vaccinia virus known as modified vaccinia virus Ankara (MVA), which carries the papillomavirus E2 gene, was used for the treatment of tumors associated with human papillomavirus. METHODS: Analysis of expression of the E2 gene from the recombinant vaccinia virus was performed by reverse transcription-polymerase chain reaction of RNA isolated from infected cells. Detection of the E2 protein was done by immunoprecipitation from proteins labeled with [(35)S]-methionine, isolated from infected cells. The therapeutic effect of the MVA E2 recombinant virus over human tumors was tested in nude mice bearing tumors generated by inoculation of HeLa cells. Series of 10 nude mice with tumors of different sizes were injected with MVA, MVA E2, or phosphate-buffered saline. Tumor size was monitored every week to assess growth. RESULTS: The MVA E2 recombinant virus efficiently expressed the E2 protein in BS-C-1 cells. This protein was able to repress, in vivo, the papillomavirus P105 promoter, which controls the expression of the E6 and E7 oncoproteins. In nude mice the MVA E2 virus reduced tumor growth very efficiently. In contrast, tumors continued to grow in mice treated with MVA or PBS. The life expectancy of MVA E2-treated mice was also increased three- to fourfold compared with that of animals that received MVA or PBS. CONCLUSIONS: The growth of human tumors was efficiently inhibited by the MVA E2 recombinant vaccinia virus. The absence of side effects in treated animals suggested that the MVA E2 virus is a safe biologic agent that could in the future be used in humans for the treatment of cervical carcinoma.

Fertility of Y(TIR).B6 sex-reversal females with XX orthotopic ovarian transplants.

When the Y chromosome of Mus musculus domesticus (Y(TIR)) was introduced onto the C57BL/6J (B6) mouse background, testis development was impaired and half of the XY progeny (Y(TIR).B6) developed a female phenotype. Y(TIR).B6 fetal ovaries showed massive death of medullary oocytes and, after birth, produced abnormal levels of steroid hormones, exhibited irregular estrous cycles, and failed to become fertile. In this study we examined whether alterations during perinatal development observed in Y(TIR).B6 ovaries permanently impaired the establishment of the hypothalamus-pituitary-ovary axis (HPOa). B6 fetal and postnatal ovaries at different stages (fetal, infantile, or adult) were transplanted orthotopically (to the ovarian bursa) to either ovariectomized B6 normal females or Y(TIR).B6 sex-reversal females. Percentage of pregnancy, litter size, and capacity to feed pups were recorded. Reciprocally, XY(TIR).B6 ovaries were orthotopically transplanted into B6 females. After crossing with fertile males, several Y(TIR).B6 sex-reversal females with B6 ovarian transplants at all ages became pregnant, had offspring, and fed their pups. On the other hand, none of the B6 female hosts with XY(TIR) ovaries became pregnant. Results demonstrated that Y(TIR).B6 sex-reversal females maintain a functional HPOa and that their failure to reproduce is primarily due to an ovarian defect.

Differential expression of SOX9 in gonads of the sea turtle Lepidochelys olivacea at male- or female-promoting temperatures.

In mouse and chick embryos, the SOX9 gene is down-regulated in genetic females whereas in genetic males it remains in the Sertoli cells. We studied the distribution of SOX9 protein in developing genital ridges of embryos of the sea turtle Lepidochelys olivacea incubated at male- or female-promoting temperatures, using the antibody for detection. At stages 22-24, cells in medullary cords show SOX9 positive nuclei, while coelomic epithelial cells appear negative. At stage 25 however, most medullary cells are SOX9 negative and at the female-promoting temperature, and from stage 26 onwards, SOX9 protein is not detected. At the male-promoting temperature, medullary cords remain SOX9-positive at all stages. These results suggest that SOX9 is up-regulated in Sertoli cells irrespective of primary sex-determining switch. Sex is irreversibly determined at stage 24 or 26 at the male- or female-promoting temperature, respectively (Merchant-Larios et al.,'97). The present results suggest that there is a correlation between SOX9 expression and sex determination in the olive ridley. At the male-promoting temperature, Sertoli cells expressing SOX9 become committed at stage 24 and male sex is determined, whereas at the female-promoting temperature, SOX9 is down-regulated at stage 26 and female sex is determined. J. Exp. Zool. 284:705-710, 1999.

Acetylcholinesterase-positive innervation is present at undifferentiated stages of the sea turtle Lepidochelis olivacea embryo gonads: implications for temperature-dependent sex determination.

In embryos of different reptile species, incubation temperature triggers a cascade of endocrine events that lead to gonad sex differentiation. The cellular and molecular mechanisms by which temperature sets in motion this process are still controversial. Here, we begin evaluating the possible participation of the nervous system in temperature-dependent sex determination by showing the existence and origin of acetylcholinesterase (AchE)-positive nerve fibers in undifferentiated gonads of the Lepidochelys olivacea (L. olivacea) sea turtle putative male and female embryos, along the thermosensitive period for sex determination (TPSD; stages 20-27). AChE-positive nerve bundles and fibers were readily visualized until developmental stage 24 and thereafter. DiI injections and confocal imaging showed that some of these gonadal nerves arise from the lower thoracic and upper lumbar spinal cord levels, and might thus be sensory in nature. Because the vertebrate spinal cord is capable of integrating by itself thermoregulatory responses with no intervention of uppermost levels of the central nervous system, we also evaluated spinal cord maturation during the TPSD. The maturation of the spinal cord was more advanced in putative female than in male embryos, when sex determination is taking place for each sex; this process starts and ends earlier in male than in female embryos. Together these observations open the possibility that the spinal cord and the innervation derived from it could play a direct role in driving or modulating the process of temperature-dependent gonad sex determination and/or differentiation, particularly in female L. olivacea embryos.