Sexing of murine and bovine embryos by developmental arrest induced by high-titer H-Y antisera

Ramalho, Michal; Garcia, J. M.; Esper, César Roberto; Vantini, Roberta; Alves, Beatriz da Costa Aguiar; Almeida, Ivo Luis; Lima, Vera Fernanda Martins Hossepian de; Moreira-Filho, Carlos Alberto

doi:10.1016/j.theriogenology.2004.03.014

Cited by 15 publications

(14 citation statements)

References 21 publications

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“…The accuracy of the XY FISH method in this study was confirmed by existing PCR techniques [15,16]. These and other PCR techniques provided rapid, reliable indication of gender in mice, and in our laboratory, have been reliable.…”

Section: Discussionsupporting

confidence: 70%

“…Eleven conceptuses from a single female were collected and divided into two portions, one for XY FISH analysis and one for sex determination PCR. Labeled cells via FISH from each conceptus (d-n) are positioned relative to the corresponding PCR sample in their respective ethidium bromide gel lane (male-specific sry PCR; Ramalho et al [16]). Conceptus cells are labeled with chromosome-specific painting DNA probes for X (FITC/green) and Y (Cy3/red).…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, such analyses can be costly for routine use where large numbers of measurements are required and have no value at early developmental stages prior to the emergence of sexually dimorphic landmarks. Detection of DNA sequences specific to X and Y chromosomes through the use of PCR has been employed frequently as a method of gender determination in isolated preimplantation or postimplantational conceptuses [14][15][16]. This technique is particularly effective for processing large numbers of samples, but the potential exists for erroneous sex identification due to selective amplification of X chromosome genes from maternal uterine tissue that inadvertently occurs in species with highly invasive placentation, such as in rodents and humans [17,18] or where preferential amplification of one allele occurs relative to the other [19].…”

Section: Introductionmentioning

confidence: 99%

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Fluorescent in situ hybridization for sex chromosome determination before and after fertilization in mice

2007

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In mice, the relative numbers of male and female pups per litter not only can vary but can probably change over the course of pregnancy in response to numerous environmental and physiological factors. As such, a technique is required to determine gender at several developmental stages. Here we describe a robust and accurate fluorescent in situ hybridization (FISH) procedure for determining chromosomal sex that can be applied with minimal modification to sperm, pre-and post-implantation conceptuses and recovered dead post-natal pups. Sperm was prepared for FISH analysis y using a modified microwave decondensation-denaturation technique. Preimplantation conceptuses (0.5 dpc) were cultured to the morula stage before sexing. They were then acid-treated to remove the zona pellucida. Tissue homogenates from postimplantational conceptuses (8.5 dpc) and stillborn pups were fixed to pre-etched slides. Specimens were hybridized with identical, commercially available DNA probes for the X (FITC) and Y (Cy3) chromosomes. Sperm ratios met the expected value of 0.5 when determined by using XY FISH. Preimplantation conceptuses pre-treated with pepsin yielded distinct fluorescence of X and Y chromosomes in morulae, whereas microwave decondensation resulted in loss of conceptuses from the slide. Both 4.0 and 8.5 dpc conceptuses displayed mean sex ratios of 0.5. Post-natal FISH analysis allowed gender identification of pups that could not be sexed due to developmental abnormalities or partial cannibalism. FISH analysis of sperm and of multiple conceptuses or post-natal tissue provided a cost-effective, accurate alternative to PCR-based sex determination.

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Section: Discussionsupporting

confidence: 70%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Fluorescent in situ hybridization for sex chromosome determination before and after fertilization in mice

2007

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“…Even the milder ABO incompatibility sometimes leads to neurological impairment (hemolysis and jaundice). The proposed anti-H-Y antigen should act in the same way, and in fact, it is used to sex, arrest development and kill male embryos (Shelton and Goldberg, 1984;White et al, 1987;Ramalho et al, 2004). In our hypothesis, H-Y, which is present from the eight-cell embryo (Blanchard, 2004;Blanchard and Klassen, 1997), and SRY (the main gene for gonad differentiation into testis), present twelve hours after fertilization (Ao, Erickson, Winston and Handyside, 1994) are good candidates to induce maternal tolerance rather than rejection.…”

Section: Discussion and The New Hypothesis Based On Fetal-maternal Tomentioning

confidence: 99%

Sexual orientation, handedness, sex ratio and fetomaternal tolerance-rejection

Valenzuela

2010

Biol. Res.

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Fraternal birth order (FBO) appears as a prenatal cause of 15% of homosexual males (gays) through mnemonic maternal anti-male factors. Non-right-handed men seem to be protected from homosexuality. Four hypotheses are proposed: (1) androgenic factors of nonright-handedness neutralize anti-male factors; (2) non-right-handedness and homosexuality are lethal or produce mental impairment; (3) non-right-handed male embryos are insensitive to anti-male factors; (4) mothers of non-right-handed fetuses do not produce antimale factors. Studies of the sex ratio (SR) of older and younger siblings show: (1) a significant heterogeneity in the SR of siblings of right or non-right handed heterosexual men and women; (2) lesbians are born among siblings with high SR; (3) siblings of righthanded gays show a higher SR than non-right-handed gays that present a low SR. Based on our discovery of maternal tolerancerejection processes, associated with genetic systems (ABO, Rh), where zygotes or embryos different from their mother induce better pregnancy and maternal tolerance than do those that share antigens with their mothers, I propose a new explanation for sexual relationships, sexual orientation, handedness and sibling SR. Lesbian embryos could induce tolerance from mothers with anti-female factors. Non-right-handedness could induce maternal tolerance, or change the maternal compatibility of "gay" embryos. Alternatively, gay embryos could be poor inducers of maternal tolerance towards male traits.Key terms: homosexuality, handedness, sex ratio, birth order, fetomoternal tolerance, genetic distortions biological brothers, reared together or apart from the index homosexual case, are responsible for FBO effect. The probability of being homosexual increases as the number of older brothers increases. Handedness interacts with homosexuality and the SR of siblings; homosexuality, due to FBO, affects non-right-handed men less (Blanchard, 2001(Blanchard, , 2004Blanchard and Bogaert, 1996;Cantor et al., 2002;Bogaert, 2003Bogaert, , 2005Bogaert, , 2006, a relationship explained hypothetically by four prenatal mechanisms Lippa, 2006, 2007;Blanchard, 2008): (1) androgenic factors associated with non-right-handedness could cancel maternal anti-male factors leading to homosexuality; (2) homosexuality and non-right-handedness together may lead to death, neuro-psychic impairment or any condition that confers a low probability of being ascertained; (3) non-right handed fetuses my be insensitive to maternal anti-male factors; (4) mothers of non-right handed fetuses do not produce anti-male factors. The FBO was found also in transsexuals whose sexual identity, rather than their sexual orientation, is altered (Green, 2000). These hypotheses are in contradiction with the fact that non-righthandedness is more frequent in homosexual men (gays) and women (lesbians) than in heterosexuals (Lalumiére et al., 2000; this study). A hypothetical reconciliation of these contradictory facts was presented by Blanchard (2008). The authors (of the FBO hypoth...

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“…One of them, the loopmediated isothermal amplification (LAMP) is less time consuming and offers quite promising results with a high accuracy (Hirayama et al, 2004). At the same time there was published the paper describing how the high-titer rat H-Y antibody induced the developmental arrest of male embryos (Ramalho et al, 2004). This non-invasive embryo sexing requires further elucidation, however.…”

Section: Discussionmentioning

confidence: 99%

Lower quality bovine embryos may be successfully used for sex determination

Lopatářová¹,

Čech²,

Krontorád³

et al. 2007

Vet. Med. - Czech

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AbstrAct:The aim of this study was to evaluate the effect of sex determination procedures in Day 7 to Day 8 bovine embryos of various quality. For the purposes of comparison we used high quality (HQ) as well as lower quality (LQ) embryos obtained from superovulated donors. The healthy embryonic cells isolated from HQ embryos and blastomeres protruding into the perivitelline space of LQ embryos, were analysed by polymerase chain reactions (PCR) using primers specific for the Y chromosome determinant. After microsurgical intervention and completion of sex determination, the female embryos were then transferred (ET) to synchronized recipients. A total of 310 embryos of HQ were used and gender was safely determined in 275 cases (88.7%). PCR analysis of extruded cells isolated from 170 LQ embryos was carried out with certainty only in 111 embryos (65.3%, P < 0.01). After ET of 122 HQ sex defined embryos, pregnancy was established in 69 recipients (56.6%). A similar conception rate 51.9% (27/52) was found after the ET of sex defined embryos designated as LQ. The accuracy of analysis was confirmed after calving and revealed that designated female sex coincided with 95.5% and 96.2% of calves when HQ and LQ embryos were transferred, respectively. Our results clearly show that a microsurgical technique in combination with PCR method represents a rapid and reliable approach for sex determination in HQ as well as in LQ preimplantation bovine embryos and can be used in field conditions for the regulation of the sex of progeny in selected herds.

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Sexing of murine and bovine embryos by developmental arrest induced by high-titer H-Y antisera

Cited by 15 publications

References 21 publications

Fluorescent in situ hybridization for sex chromosome determination before and after fertilization in mice

Fluorescent in situ hybridization for sex chromosome determination before and after fertilization in mice

Sexual orientation, handedness, sex ratio and fetomaternal tolerance-rejection

Lower quality bovine embryos may be successfully used for sex determination

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