KR101545978B1 - Method for preponderance of female offspring - Google Patents
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Abstract
본 발명은 (a) 포유동물의 수컷 종으로부터 정자를 수집하는 단계; (b) 상기 정자를 pH 4.5 내지 5.5 조건 하에서 처리하는 단계; 및 (c) 상기 조건 처리 후 생존한 정자를 상기 포유동물의 난자에 주입하여 인공수정시키는 단계를 포함하는 암컷 수태율 증진방법에 관한 것이다. 본 발명의 암컷 수태율 증진방법에 따르면 포유동물의 암컷 수태율을 증가시키기 위하여 숙련된 기술이나 고가의 장비를 사용이 필요하지 않으며, 정자에 심각한 손상 없이 안전하게 암컷 수태율을 증가시킬 수 있다.(A) collecting sperm from a male species of a mammal; (b) treating the sperm under pH 4.5 to 5.5 conditions; And (c) injecting the sperm survived after the condition treatment into the oocyte of the mammal, thereby artificially correcting the female conception rate. According to the method of increasing the female conception rate according to the present invention, it is not necessary to use a skilled technique or expensive equipment to increase the female conception rate of the mammal, and the female conception rate can be safely increased without serious damage to the sperm.
Description
본 발명은 포유동물의 암컷 수태율을 증진시키는 방법에 관한 것이다.The present invention relates to a method for enhancing the female conception rate of a mammal.
기존의 정자의 성을 분류하는 방법으로서 FACS (Fluorescence Activated Cell Sorting)는 X염색체와 Y염색체의 DNA량의 차이에 따라 정자를 분리해 낼 수 있다. FACS 분석법은 기계를 사용하기 때문에 많은 양의 정자를 분석할 수 있으며 그 유효성이 입증된 바 있다 (Cordelli E. 2005). 그러나 1991년 이미 X-, Y- 정자의 DNA량의 차이에 따른 FACS 분류 방법은 L.A.Johnson에 의해 특허가 출원되어 그 이용방법에 다소 제한이 따른다. 이러한 제한 외에도 FACS 분류기를 통과하는 동안 염색, 레이저에 의한 노출, 분리액에 의한 고농도의 희석, 압력, 분류과정에 있어서 사용되어 배양액의 조성변화 등의 다양한 기계적 정자 손상 요인이 존재하고 있어 FACS 분류기를 통과한 대부분의 정자는 꼬리가 잘려나가거나 핵을 둘러싼 세포막에 심각한 손상을 받는다 (Maxwell 1999; Guthrie 2002; Vazquez 2002; Seidel 2003).FACS (Fluorescence Activated Cell Sorting) can separate spermatozoa according to the amount of DNA of X chromosome and Y chromosome. Since the FACS method uses a machine, a large amount of sperm can be analyzed and its effectiveness has been proven (Cordelli E. 2005). However, the FACS classification method according to the difference in DNA amount of X- and Y-spermatozoa was already filed by L.A. Johnson in 1991, and its usage method is somewhat limited. In addition to these limitations, there are various mechanical sperm damage factors such as dyeing, laser exposure, dilution at high concentration by separation solution, pressure, classification of culture medium, etc. during passage through the FACS classifier. Most of the sperm that pass through are severely damaged by the tail cut off or the cell membrane surrounding the nucleus (Maxwell 1999; Guthrie 2002; Vazquez 2002; Seidel 2003).
한편, FISH (fluorescence in situ hybridization)나 CISH (chromogenic in situ hybridization)와 같은 ISH (in situ hybridization)분석은 태그 (tag)나 라벨 (label)을 포함하고 있는 프로브 (probe)를 표적 염색체의 특정 뉴클레오티드 서열에 상보적으로 결합시켜 유전자의 존재 여부를 결정하는데 사용된다. ISH 분석을 통한 성 선별은, FACS로 인한 정자의 손상을 막아 수정능과 같은 생물학적 활성을 높여, 현재 많이 사용되고 있는 기술 중 하나이다. 다만, 이러한 기술은 성 선별을 위해 일정 단계의 숙련된 기술을 필요로 한다.On the other hand, in situ hybridization (ISH) analysis such as fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) is a technique in which a probe containing a tag or a label is inserted into a specific nucleotide Are used to determine the presence or absence of a gene by complementarily binding to a sequence. Sex screening through ISH analysis is one of the technologies currently used, since it inhibits sperm damage caused by FACS and improves biological activity such as fertility. However, these techniques require a certain level of skill to select sex.
따라서 기존의 정자의 성 선별 방법들에 의할 경우, 성 성별된 정자에 심각한 손상을 주는 점 및 실험이 복잡하여 숙련된 기술을 요하고 고가의 장비를 사용해야 한다는 점 등의 문제점이 여전히 존재한다.Therefore, there is still a problem in that the sperm sex screening methods of the existing sperm severely damage spermatozoa sperm and require expensive techniques because of complex experimentation and expensive equipment.
이에 본 발명자들은 상기의 문제점을 극복하기 위하여 여러 가지 조건하에서 X-정자 및 Y-정자의 선별을 연구한 결과, 특정 pH조건 하에서 정자를 처리할 경우, 복잡한 성 선별 방법을 통하지 않고도 X-정자의 상대적 비율이 증대됨을 확인함으로써 본 발명을 완성하였다.As a result of studying the selection of X-spermatozoa and Y-spermatozoa under various conditions in order to overcome the above problems, the present inventors have found that when spermatozoa are treated under specific pH conditions, And the relative ratio is increased, thereby completing the present invention.
본 발명은 The present invention
(a) 포유동물의 수컷 종으로부터 정자를 수집하는 단계; (a) collecting sperm from a male species of a mammal;
(b) 상기 정자를 pH 4.5 내지 5.5 조건 하에서 처리하는 단계; 및(b) treating the sperm under pH 4.5 to 5.5 conditions; And
(c) 상기 조건 처리 후 생존한 정자를 상기 포유동물의 난자에 주입하여 인공수정시키는 단계를 포함하는 암컷 수태율 증진방법을 제공한다.(c) injecting the surviving sperm into the oocyte of the mammal after the condition treatment, thereby artificially correcting the female conception rate.
본 발명의 암컷 수태율 증진방법에 따르면 포유동물의 암컷 수태율을 증가시키기 위하여 숙련된 기술이나 고가의 장비를 사용이 필요하지 않으며, 정자에 심각한 손상 없이 안전하게 암컷 수태율을 증가시킬 수 있다.According to the method of increasing the female conception rate according to the present invention, it is not necessary to use a skilled technique or expensive equipment to increase the female conception rate of the mammal, and the female conception rate can be safely increased without serious damage to the sperm.
도 1은 배지 pH 조건 (control: pH 7.0, pH 4.5, pH 5.5)에 따른 정자 생존율을 측정한 그래프이다.
도 2는 낮은 pH 조건 (pH 4.5, pH 5.5)하에서 30분 동안 정자를 배양한 경우 전체 정자들을 기준으로 X-정자의 상대적 증가 비율을 나타낸 그래프이다.FIG. 1 is a graph showing sperm survival rate according to medium pH conditions (control: pH 7.0, pH 4.5, pH 5.5).
FIG. 2 is a graph showing the relative increase rate of X-spermatozoa based on total spermatozoa cultured for 30 minutes under low pH conditions (pH 4.5, pH 5.5).
이하 본 발명을 자세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명은 The present invention
(a) 포유동물의 수컷 종으로부터 정자를 수집하는 단계; 및(a) collecting sperm from a male species of a mammal; And
(b) 상기 정자를 pH 4.5 내지 5.5 조건 하에서 처리하는 단계; 및(b) treating the sperm under pH 4.5 to 5.5 conditions; And
(c) 상기 조건 처리 후 생존한 정자를 상기 포유동물의 난자에 주입하여 인공수정시키는 단계를 포함하는 암컷 수태율 증진방법을 제공한다.(c) injecting the surviving sperm into the oocyte of the mammal after the condition treatment, thereby artificially correcting the female conception rate.
본 발명에 따른 상기 (a) 단계에서, 포유동물은 포유동물에 해당하면 이를 특별히 제한하는 것은 아니나, 돼지, 소, 양 및 말 등이 바람직하다.In the step (a) according to the present invention, when the mammal is a mammal, it is not particularly limited, but pigs, cows, sheep and horses are preferred.
본 발명에 따른 상기 (b) 단계에서, 정자는 pH 4.5 내지 5.5 조건 하에서 1분 내지 60분간 처리하는 것이 좋다. 처리하는 시간이 60분을 초과할 경우 정자의 이수성이 나타날 수 있다. 또한, 상기 정자는 복수의 정자를 대상으로 함으로써 수태율을 증대시킬 수 있다. In the step (b) according to the present invention, the spermatozoa are preferably treated at pH 4.5 to 5.5 for 1 minute to 60 minutes. If the treatment time exceeds 60 minutes, the sperm integrity may appear. In addition, the sperm can increase the pregnancy rate by targeting a plurality of sperm.
본 발명에 따라 정자를 pH 4.5 내지 5.5 조건 하에서 1분 내지 60분간 처리할 경우, Y-정자의 생존율이 급격히 떨어지고 이로 인하여 생존된 정자는 X-정자의 비율이 상대적으로 증가되게 된다.According to the present invention, when the sperm is treated for 1 minute to 60 minutes under the condition of pH 4.5 to 5.5, the survival rate of Y-sperm rapidly decreases and the proportion of X-sperm in the survived sperm is relatively increased.
본 발명에 따른 상기 (c) 단계는 상기 (b) 단계의 조건 처리 후 생존한 정자를 상기 포유동물의 난자에 주입하여 인공수정시키는 단계이다. 또한, 상기 단계에서 포유동물 난자의 세포질 내에 주입하는 것이 바람직하다. 상기 (b) 단계의 조건 처리 후 생존한 정자는, X-정자의 비율이 상대적으로 증가되기 때문에 상기 (b) 단계의 조건 처리 후 생존한 정자를 상기 포유동물의 난자의 세포질 내에 주입하여 인공수정시키는 경우 암컷 수태율 증진되게 된다.
In the step (c) according to the present invention, the sperm surviving after the conditioning of the step (b) is injected into the oocyte of the mammal for artificial insemination. It is also preferred to inject into the cytoplasm of the mammalian oocyte in this step. Since the ratio of X-sperm is relatively increased, the sperm surviving after the conditioning treatment in the step (b) is injected into the cytoplasm of the oocyte of the mammal after the conditioning treatment of the step (b) The conception rate of the female is increased.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어 있는 실시예를 참조하면 명확해질 것이다. 그러나 본 발명은 이하에서 개시되는 실시예에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 본 실시예는 본 발명의 개시가 완전하도록 하고, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다.
Advantages and features of the present invention, and methods of accomplishing the same, will be apparent from and elucidated with reference to the embodiments described hereinafter in detail. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art. To fully disclose the scope of the invention to a person skilled in the art, and the invention is only defined by the scope of the claims.
[[ 실시예Example ]]
실시예Example 1- One- pHpH 조건에 따른 X-, Y-정자의 생존율 측정Measure the survival rate of X- and Y-spermatozoa according to the conditions
실시예1Example 1 -1: 배지 준비-1: Prepare the badge
정자의 배양을 위하여 변형된 HEPES로 완충된 Tyrod's-Albumin-Lactate-Pyruvate 배지 (이하 간편하게 ‘mHTALP’라 함)를 제조하였다. 본 배지는 114mM의 염화나트륨, 0.3mM의 제1인산나트륨 (NaH2PO4), 3.1mM의 염화칼슘, 0.4mM의 염화마그네슘, 10mM의 젖산나트륨 (sodium lactate), 2mM의 탄산수소나트륨 (NaHCO3), 0.2mM의 sodium pyruvate, 10mM의 HEPES 및 10μg/mL의 젠타마이신으로 구성되었다. 또한 mHTALP 배지에 정자의 배양을 위하여 0.3% BSA을 첨가하였다. 배지의 pH는 염산과 수산화나트륨을 이용하여 pH 4.5 및 pH 5.5를 조정하였으며, 각각의 pH별 배지의 안정화를 위하여 22℃에서 5일간 예비 테스트를 실시하였다.
Tyrod's-Albumin-Lactate-Pyruvate medium (hereinafter simply referred to as 'mHTALP') buffered with modified HEPES was prepared for sperm culture. The medium contained 114 mM sodium chloride, 0.3 mM sodium phosphate monobasic (NaH2PO4), 3.1 mM calcium chloride, 0.4 mM magnesium chloride, 10 mM sodium lactate, 2 mM sodium hydrogencarbonate (NaHCO3), 0.2 mM sodium pyruvate, 10 mM HEPES and 10 ug / mL of gentamicin. In addition, 0.3% BSA was added to mHTALP medium for culturing sperm. The pH of the culture medium was adjusted to pH 4.5 and pH 5.5 with hydrochloric acid and sodium hydroxide, and preliminary tests were conducted at 22 ° C for 5 days to stabilize the media at each pH.
실시예1Example 1 -2: 정액샘플 준비-2: Preparation of semen sample
정액 샘플은 세계보건기구 표준에 따라 3일간 금욕한 11명의 정상적으로 정자 생산 가능한 기증자들로부터 자위를 통하여 얻을 수 있었다. 이러한 정자 샘플의 특성은 다음과 같았다. 용량은 2.35±0.23mL, 농도는 94.54±11.62×106/mL, 운동성은 72.57±5.08%, 기타 세계보건기구 표준에 따른 정자 형태를 확인할 수 있었다. Sperm samples were obtained through masturbation from eleven normally sperm-producers who were abstinent for three days according to World Health Organization standards. The characteristics of this sperm sample were as follows. The dose was 2.35 ± 0.23 mL, the concentration was 94.54 ± 11.62 × 10 6 / mL, the motility was 72.57 ± 5.08%, and other forms of spermatozoa according to the World Health Organization standards were confirmed.
정자 샘플의 적절한 양의 사용을 위하여, 4종류의 정자 샘플을 정상 남자로부터 얻은 후 4종류의 샘플들을 모두 혼합하였다. 정장을 제거하기 위하여 혼합된 샘플은 mHTALP 배지로 희석된 후, 5분 동안 400×g로 원심분리하였다. 상등액은 조심스럽게 제거하고 침전물은 5mL mHTALP 배지로 2번 세척하였다. 최종 정자 농도는 0.3% BSA을 함유한 mHTALP 배지 안에 1×106/mL가 되도록 조정하였다.
For the proper use of sperm samples, four sperm samples were obtained from normal men and all four samples were mixed. Mixed samples were diluted with mHTALP medium to remove formalin and then centrifuged at 400 x g for 5 minutes. The supernatant was carefully removed and the precipitate was washed twice with 5 mL mHTALP medium. The final sperm concentration was adjusted to 1 x 10 6 / mL in mHTALP medium containing 0.3% BSA.
실시예1Example 1 -3: 정자 배양-3: sperm culture
상기 실시예1-2에 따라 최종 정자 농도가 조정된 0.3% BSA를 함유한 mHTALP 배지 5mL는 22℃에서 60분간 인큐베이션되었다.
5 mL of the mHTALP medium containing 0.3% BSA in which the final sperm concentration was adjusted according to Example 1-2 was incubated at 22 DEG C for 60 minutes.
실시예1Example 1 -4: 저장액팽창검사 (-4: Storage liquid expansion test ( hypoosmotichypoosmotic swellingswelling testtest : : HOSTHOST ) 및 ) And 정자핵의Sperm nuclear 전처리 Pretreatment
정자의 배지 pH 조건에 따른 생존율을 측정하기 위하여 저장액팽창검사를 실시하였다.Storage fluid dilatation test was performed to measure the survival rate according to the pH condition of sperm medium.
저장성 용액의 용적의 절반은 milli-Q water를 포함하고, 용적의 다른 절반은 기본 배지로 조성하였다. 최종 삼투압농도가 140mosmol/kg가 될 수 있도록 제조하였다. 1mL 37℃ 저장성 용액에 정액을 떨어뜨린 후, 37℃ 챔버에서 5분 동안 인큐베이션시켰다. 인큐베이션 후에 정자 및 저장성 용액 혼합물을 원심분리하고, 원심분리에 따른 침전물은 100-200ml 저장성 용액 안에서 다시 희석되었다. 상기 서스펜션을 깨끗한 슬라이드 상에 도말한 후 공기 중에 건조시키고, 접착액으로 고정화시켰다. 건조된 슬라이드는 37℃에서 45분간 2mmol/L 디티오트레이톨 (dithiothreitol) 안에서 인큐베이션시킨 후 실내에서 건조하였다. 상기 건조된 슬라이드는 70% formamide/2XSSC 용액에 5분 동안 변성화시켰다. 형광 현미경 하에서 정자 꼬리 팽창 패턴을 평가하기 위하여, 변성된 견본(표본)을 Diff Quick (Baxter, Deerfield,IL)를 사용하여 염색하고, 50%, 70%, 95%의 에탄올로 실내 온도에서 3분 동안 탈수한 후, 공기 건조하였다.Half of the volume of the storage solution contained milli-Q water, and the other half of the volume was composed of the basal medium. The final osmotic concentration was made to be 140 mosmol / kg. Sperm was dropped in 1 ml 37 ° C stock solution and incubated in a 37 ° C chamber for 5 minutes. After incubation, the sperm and the stock solution mixture were centrifuged, and the precipitate from centrifugation was diluted again in 100-200 ml of storage solution. The suspension was plated on a clean slide, dried in air, and fixed with an adhesive solution. The dried slides were incubated in 2 mmol / L dithiothreitol at 37 < 0 > C for 45 minutes and then dried indoors. The dried slides were denatured in 70% formamide / 2X SSC solution for 5 minutes. To evaluate the sperm tail expansion pattern under fluorescence microscopy, the denatured specimens were stained with Diff Quick (Baxter, Deerfield, Ill.) And incubated with 50%, 70%, 95% Deg.] C, and air dried.
그 결과 도 1에서 나타낸 바와 같이, 정자 생존율은 시간 의존적으로 pH 4.5 and pH 5.5 배지에서 급격히 줄어드는 것이 관찰되었다.
As a result, as shown in Fig. 1, the sperm survival rate was observed to decrease sharply at pH 4.5 and pH 5.5 in a time-dependent manner.
실시예1Example 1 -5: 형광면역접합법 (-5: Fluorescence immunoassay ( fluorescence여광체 inin situsitu hybridizationhybridization : : FISHFISH ))
배지 pH 조건에 따른 X- 및 Y- 정자의 생존율 차이를 측정하기 위하여 상기 저장액팽창검사를 통해 확보된 생존 정자들을 이용하여 형광면역접합법을 실시하였다.To measure the difference in the viability of X- and Y-spermatozoa according to the pH conditions of the medium, fluorescence immunoassay was performed using viable sperm obtained through the above-mentioned storage fluid expansion test.
형광면역접합법을 실행하기 위하여 표적에 부착되는 탐침 (probe)은 염색체 X, Y 및 18의 특이적 알파 위성 동원체 탐침이 사용되었다. 이들 탐침은 플라티넘 브라이트 550 (Red signal; Kreatech, Amsterdam, Netherland) (chromosome X, CEP X; chromosome 18, CEP 18) 또는 플라티넘 브라이트 495 (Green signal; Kreatech, Amsterdam, Netherland) (chromosome Y, CEP Y; chromosome 18, CEP 18)로 직접적으로 라벨링되었다. 또한 yellow signal은 플라티넘 브라이트 550로 라벨링된 염색체 18 탐침과 플라티넘 브라이트 495로 라벨링된 염색체 18 탐침들의 동일 용량의 혼합으로 얻을 수 있었다.Specific alpha satellite probe of chromosomes X, Y, and 18 was used as a probe attached to the target to perform fluorescent immunoassay. These probes are available from Plasmal Bright 550 (Red signal; Kreatech, Amsterdam, Netherland) (chromosome X, CEP X, CEP 18) or Platinum Bright 495 Y; chromosome 18, CEP 18). The yellow signal was also obtained by mixing the same volume of chromosome 18 probes labeled with PlatinumBlue 550 and chromosome 18 probes labeled with PlatinumBlue 495.
혼성화 혼합물은 3종류의 탐침들 각각 1μL씩, 6μL의 포름아마이드, 1μL의 20×SSC를 포함하여 총 용량이 10μL가 되도록 하였다. 42℃로 온도를 맞춰둔 (prewarmed) 슬라이드 상에 상기 혼성화 혼합물을 첨가한 후, 22×22mm 커버슬립으로 덮었다. 이렇게 준비된 슬라이드는 80℃에서 5분 동안 변성되었다. 그리고 모든 슬라이드들은 42℃ 습윤 챔버에서 16시간 동안 혼성화되었다. 전 혼성화 커버슬립은 50% 포름아마이드/2×SSC 안에서 슬라이드로부터 분리되었으며, 37℃에서 pH 7.0의 50% 포름아마이드/2×SSC로 3번 세척된 후, pH 7.0의 0.1% NT40/2×SSC로 5분 동안 세척되었다. 상기 과정을 통한 슬라이드들은 실온에서 5분 동안 PBS 안에서 헹구었다. The hybridization mixture contained 1 μL of each of the three probes, 6 μL of formamide, and 1 μL of 20 × SSC to a total volume of 10 μL. The hybridization mixture was added onto a prewarmed slide at 42 ° C and then covered with 22 × 22 mm cover slip. The slide thus prepared was denatured at 80 DEG C for 5 minutes. And all the slides were hybridized for 16 hours in a 42 ° C humid chamber. The prehybridized coverslip was separated from the slides in 50% formamide / 2 x SSC and washed three times with 50% formamide / 2 x SSC at pH 7.0 at 37 占 then resuspended in 0.1% NT40 / 2 占 SSC For 5 minutes. Slides through the process were rinsed in PBS for 5 minutes at room temperature.
상기 슬라이드들은 epi-fluorescence illumination 하에 Nikon microphot-FXA로 점수를 매겼다. 주관적인 판결을 방지하기 위하여 슬라이드는 한 사람이 라벨화하고, 다른 사람이 점수를 매기는 형식으로 하였다.The slides were scored with Nikon microphot-FXA under epi-fluorescence illumination. To prevent subjective judgments, slides were labeled by one person and scored by others.
통계분석은 통계소프트프로그램 (SPSS Version 12.0, USA)을 사용하여 수행되었다. X- 및 Y- 정자 사이의 생존률은 t-test에 의해 비교되었다. X- 및 Y- 정자의 생존 차이는 반복되는 ANOVA 측정에 의해 분석되었다. Statistical analysis was performed using the Statistical Soft Program (SPSS Version 12.0, USA). Survival rates between X- and Y-sperm were compared by t-test. The survival difference of X- and Y-sperm was analyzed by repeated ANOVA measurements.
그 결과 도 2에서 나타낸 바와 같이, pH 4.5 and pH 5.5 조건 하에서 X 정자의 생존율이 Y정자의 생존률보다 높은 것을 알 수 있었다.As a result, as shown in FIG. 2, the survival rate of X sperm was higher than that of Y sperm at pH 4.5 and pH 5.5.
Claims (6)
(b) 상기 처리에 의해 생존한 정자를 포유동물(단, 인간은 제외) 유래 난자의 세포질 내에 주입하여 인공수정시키는 단계를 포함하는 암컷 수태율 증진방법.
(a) treating sperm collected from a male species of a mammal (but not a human) under conditions of pH 4.5 to 5.5 for 1 minute to 60 minutes; And
(b) injecting the sperm survived by the treatment into the cytoplasm of the oocyte derived from a mammal (except for a human), and subjecting the fertilized egg to artificial fertilization.
2. The method of claim 1, wherein the mammal is selected from the group consisting of pigs, cows, sheep and horses.
The method according to claim 1, wherein the step (a) is performed in a mHTALP (Tyrod's-Albumin-Lactate-Pyruvate) medium.
(Y-sperm) containing Y-chromosomes containing a step of treating the sperm collected from male species of mammals (except for humans) under the condition of pH 4.5 to 5.5 for 1 minute to 60 minutes, A method for increasing the relative proportion of sperm containing a chromosome (X-sperm).
5. The method according to claim 4, wherein the mammal is selected from the group consisting of pigs, cows, sheep and horses. The spermatozoa containing the X-chromosome (Y-sperm) ) Relative to each other.
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