Norma Moreno-Mendoza

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.

Response of diencephalon but not the gonad to female-promoting temperature with elevated estradiol levels in the sea turtle Lepidochelys olivacea

Although temperature sex determination is well known in several reptile species, the physiological mechanism underlying this process remains to be elucidated. In the current work, we analyzed the levels of testosterone (T) and estradiol (E2) in the gonads; two brain regions--telencephalon (Te) and diencephalon/mesencephalon (Di)--and the serum of developing embryos of the olive ridley Lepidochelys olivacea incubated at male- or female-promoting temperatures. Conversion of pregnenolone (P5) to T and T to E2 were studied in the gonads and brain. The analyses were performed during three periods: the thermosensitive period (TSP), histologically undifferentiated gonads (UDG), and differentiated gonads (DG). In the gonads, serum, and brain, T concentrations were higher at the female-promoting temperature during the three periods, whereas in the gonads and serum, E2 levels were similar at the female and male-promoting temperature. In Di, the concentration of E2 was significantly higher at the female-promoting temperature. Biotransformation of P5 to T in gonadal tissues were slightly higher at the female-promoting temperature in TSP and increased during UDG and DG. Conversion of T to E2, however, was similar at the two temperatures during the three periods. In the brain, the Di showed a higher efficiency for transforming T to E2 at the female-promoting temperature. Our present results do not allow us to decide whether the diencephalon is the cause or the effect, but they conclusively demonstrate that, in L. olivacea, this region of the brain senses temperature during sex determination.

Effect of allopurinol on damage caused by free radicals to cryptorchid testes.

Cryptorchidism causes apoptosis of germ cells. It has been suggested that the redox regulatory system is involved in this process. The free radicals produced are thought to be generated during the production of uric acid, a reaction catalyzed by xanthine oxidase. This enzyme is inhibited by allopurinol; however, the role of allopurinol in neonate rats with inguinal cryptorchidism has not been assessed yet. Sixty male Wistar rats were used and five groups were formed: a control, a sham, a sham group with allopurinol administration and two groups with surgical unilateral cryptorchidism, which either did not receive, or received, allopurinol. The rats were assessed at 40 days post-partum. Reactive oxygen species concentration and epithelial area were measured and the histopathological, apoptotic and cellular proliferation indexes were determined. We found a decrease in reactive oxygen species, histopathological and apoptotic indexes and an increase in proliferation index and epithelial area in rats with cryptorchidism treated with allopurinol in comparison with rats with untreated cryptorchidism. We suggest that the over-production of reactive oxygen species plays an important role in the damage of the cryptorchid testes. Allopurinol administration decreases reactive oxygen species concentrations as well as the damage to the germ epithelium.

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. J. Comp. Neurol. 410:90–98, 1999.

Disturbed Expression of Sox9 in Pre-Sertoli Cells Underlies Sex-Reversal in Mice B6.Ytir

Abstract Sry in some varieties of Mus musculus domesticus fails to form normal testis when introduced into the C57BL/6J (B6) strain. We studied the developmental pattern of pre-Sertoli cells that express Sox9 by immunofluorescence and the profile levels of Sox9 transcripts by semiquantitative reverse transcriptase polymerase chain reaction and in situ hybridization in developing gonads of B6-Ytir mice. Sox9-positive cells (pre-Sertoli cells) appeared in all B6.Ytir genital ridges at 11.5 and 12.5 days postcoitum (dpc). However, at 13.5 dpc, Sox9-positive cells were not detected only in 50% of the B6.Ytir gonads compared with 100% of B6 gonads. Although pre-Sertoli cells formed the seminiferous cords after 14.5 dpc in the medial region of the B6.Ytir gonad, the cranial and caudal regions formed ovarian tissue. Further, B6.Ytir ovaries have lower levels of Sox9 than ovotestes at all fetal stages. These results suggest that although the pre-Sertoli cell lineage forms in B6.Ytir genital ridges, its further differentiation into Sertoli cells is apparently prevented. The cause may be the low levels of Sox9 and down-regulation of its product. Results suggest that inhibitory signals of Sox9 acting along the whole genital ridge or only at its cranial and/or caudal regions underlie formation of B6.Ytir ovaries or ovotestes, respectively. Furthermore, our results suggest that infertility of B6.Ytir females may be due to the abnormal presence of Sox9 transcripts in their ovaries.

RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle.

The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination.

Identification of cortical germ cells in adult ovaries from three phyllostomid bats: Artibeus jamaicensis, Glossophaga soricina and Sturnira lilium

It is generally considered that, in mammals, the ovary is endowed with a finite number of oocytes at the time of birth. However, studies concerning rodents, lemurs and humans suggest the existence of stem cells from the germline that may be involved in germ-cell renewal, maintaining postnatal follicle development. This type of work on wild species is scarce; therefore the objective of this study was to determine ovarian morphology and the presence of progenitor cells from the germline of three species of phyllostomid bats (Artibeus jamaicensis, Glossophaga soricina and Sturnira lilium). The morphological characteristics of the ovaries and the expression of specific markers of germline cells, stem cells and proliferation cells were analysed. The morphology of the ovaries of the three bat species was similar. A polarised ovary with follicles at different stages of development and groups of cortical cells similar to primordial germ cells were observed. Immunofluorescent analysis showed that these cortical cells express germline, stem-cell and proliferative markers, indicating the identification of germ cells that could maintain pluripotency, as well as being mitotically active. This suggests that in the adult ovary of phyllostomid bats there may be a mechanism for the self-renewal of the germline.

Expression of 3β-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 3β-Hsd1 and P450arom as well as protein expression pattern of 3β-HSD1 and Testosterone in normal undifferentiated and differentiated gonads from both male and female mice embryo. Our data indicate that there is expression of 3β-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 3β-Hsd1 in the early stages of ovarian development, as well as the absence of Testosterone suggests that XX gonads are not steroidogenic and that 3β-Hsd1 enzyme may play a different role than in the steroidogenesis process.

Androgen Receptor and Calcitonin Gene-Related Peptide in Neurons of the Genitofemoral Nerve During Testicular Descent Induced with Human Chorionic Gonadotropin

BACKGROUND Low levels of circulating testosterone during testis descent cause cryptorchidism in humans and rats. Treatment with human chorionic gonadotrophin (hCG) induces testis descent by stimulating production of testosterone (T). Neurons of genitofemoral nerve (GFN), which innervate testicular gubernaculum, may play a role in testis descent. METHODS In the current study, putative correlations were made between T and GFN motor and sensory neuron activity during inguinoscrotal testis descent. Cryptorchidism was provoked in prepuberal rats with estradiol. Rats with testicular descent induced with hCG and cryptorchid controls were used. Cells of spinal cord and dorsal root ganglia were labeled by retrograde staining with fast-blue. Expression of androgen receptor (AR) and calcitonin gene-related peptide (CGRP) were detected with indirect immunofluorescence. RESULTS Neurons labeled with fast-blue were found in the center of motor horn and dorsal root ganglia at levels L1 and L2. While number of motor neurons expressing AR was significantly higher in the group treated with hCG, number expressing CGRP was higher in controls. In dorsal root ganglion, number of cells immunostained with CGRP antibody was similar in both groups but AR was not detected. CONCLUSIONS Present results support the hypothesis that motor nucleus of the GFN is a direct target of testosterone and that regulation of CGRP in sensory nucleus may be involved in testicular descent.

Characterization of gametes in two phyllostomid bat species: Artibeus jamaicensis and Sturnira lilium

Morphology of gametes is used to understand the physiological processes in reproduction among domestic and wild animals. These gametes are used in assisted reproductive technology (ART) and conservation programs. In the case of Artibeus jamaicensis and Sturnira lilium, few studies have been conducted related to these issues. The aim of this study was to describe the structure of spermatozoa, semen characteristics and also the morphology and quality of cumulus oocyte complexes (COC) of A. jamaicensis and S. lilium. Semen characteristics were: A. jamaicensis had a sperm concentration of 4.26×10(6)sperm/ml, progressive motility of 34.55%; viability of 73.23%; head, tail and mid-piece abnormalities of 12.50%. Head length was 6.26μm, mid-piece 18.61μm and tail 70.92μm. S. lilium, had a sperm concentration of 5.15×10(6)sperm/ml, progressive motility of 60.00%, viability of 83.82%; abnormalities in head, tail and mid-piece of 13.77%. Head length was 7.01μm, mid-piece 20.33μm and tail 70.50μm. On average 12.8 of right ovarian oocytes and 9.9 of left ovarian oocytes of A. jamaicensis were recovered. For S. lilium on average 10.7 oocytes from the right ovary and 10.9 oocytes from the left ovary were recovered, ranging in quality from excellent to poor. Sperm morphology and quality of COC were similar to those for other domestic and wild animals. Bat gametes can be used for the study of reproductive biology, in conservation programs and assisted reproductive technology (ART) among domestic and wild animals.