Investigation on mechanism of sterility of male hybrids between yak and cattle

Abstract

Cattle–yak, the first filial generation of yak and ordinary cattle, showed obvious hybrid vigor, but a difficult problem in crossbreeding and improvement of yak is that the males are sterile. Thus it is not possible to steadily utilize the heterosis. So the study of the mechanism of male sterility is still one of the most interesting fields in yak science. Therefore, this paper will review contemporary studies in cattle–yak male sterility including the process of spermatogenesis, development of genital organs, pituitary and endocrinology, biochemical genetics and candidate genes correlating spermatogenesis, etc.

Keywords:

• cattle–yak
• male sterility
• spermatogenesis
• candidate genes

1. Introduction

Although hybridization of yak (Bos grunniens) with cattle (Bos taurus) has been used in improving yak's production for a long time, hypothesis of the association of yak with domestic cattle was erroneous. Nuclear and fossils data analyses showed that the clade uniting bison and yak is robust and reliable than cattle (Hassanin et al. 2013). And the divergence times of yak with cattle were estimated using the genetic distance derived from microsatellite markers to have diverged 0.57–1.53 million years ago (Ritz et al. 2000). In China, the earliest practice of hybridization between yak and local cattle is thought to have started during the Yin Dynasty (approximately 1100 B.C.) (Cai 1989; Zhang 2000). In hybridization of yak and ordinary cattle, the first filial generation (F1, cattle–yak) showed obvious hybrid vigor, but the male cattle–yak, although with normal secondary sexuality, is sterile because a disorder of its sperm producing function due to the effect of interspecific hereditary isolation. The female F1 shows normal genital capacity. This phenomenon was in line with Haldane's Rule, Which formulated by J. B. S. Haldane in 1922, described as: “When in the F1 offspring of two different animal races one sex is absent, rare, or sterile, that sex is the heterozygous [heterogametic] sex” (Coyne 1985; Haldane 1922). The “Haldane Rule” is followed perfectly by the cattle × yak and cattle × bison hybrid; namely, sterility is confined to the male (Baranov & Zakharov 1997; Kh et al. 2008). So for practical production there is only implemental upgrading using the parental bull, but the adaptability of the offspring declines (back-cross with fine yellow cattle bull), or its hybrid vigor disappears (back-cross with yak bull) along with the increase of hybridization generation. The sterility of the male cattle–yak has been the barrier to improving and breeding yaks.

Many studies have been done on the sterility of the male cattle–yak both at home and abroad. China was in the forefront of the world in the research fields involving hybrid combination, cytogenetics, tissue morphology, endocrinology, and biochemistry, etc. That research revealed some differences among cattle–yak with both of its parents in some subjects, which accumulated fruitful information for further study. Until now, there has been no solution to the problem of male sterility. The following is a summary of studies on hybridization between yak and cattle.

2. Study of the sperm occurring level

The testis of male hybrid offspring from hybridization of yak gradually recovered its capacity for sperm production along with the increase in upgraded hybridization generation, which indicates a gradual development in male fertility. Jia (2001) reported that in F1 male hybrid sperm producing function had been totally destroyed with no sperm in semen; F2 male hybrid had recovered its sperm producing function to some degree and occasionally found a few dead sperm in semen, but most of the live ones were in pathological condition; F3 male hybrid had greatly increased its fertility and could produce some sperm with normal morphology; almost of all F4 male hybrid had recovered the function in sperm production.

3. Study of in vitro fertilization between cattle and yak or bison

In vitro research found that B. taurus (cattle) oocytes can be fertilized with sperm from either yaks (B. grunniens) or bison (Zi et al. 2008; Seaby et al. 2012). The percentage of oocytes penetrated and the formation of two pronuclei when cattle oocytes were inseminated with yak spermatozoa (97.4 and 81.6%, respectively) were greater (p < 0.01) than that achieved with Holstein bull spermatozoa (77.8% and 65.9%, respectively). The yak ♂× cattle combination gave a higher cleavage rate than the Holstein ♂× cattle combination (p < 0.05; 76.3% vs. 63.3%) (Zi et al. 2008). Although decreased developmental competence in the wood bison hybrid embryos has been found, cattle oocytes also can be fertilized with sperm from bison. The significantly lower cell numbers than cattle blastocysts owing to inadequate culture conditions have increased apoptotic events (Seaby et al. 2012).

4. Study on somatic chromosome

Most studies have showed that cattle–yak have the same chromosome number as both parents (2n = 60), but the chromosome morphology differs. Guo (1983) found the sex chromosome of yellow cattle–yak (cattle × yak) and both of its parents were all sub-middle centromere, and Y chromosome of yellow cattle–yak and its male parent(yak) were sub-middle centromere (yellow cattle was middle centromere). The relative length showed no significant difference among the yellow cattle–yak and both parents. By studying on the chromosome of black cattle–yak (Holstein × yak) and that of the second filial generation (F2), Chen (1990) found that filial generation's autosome was the same as both parents and was near-end centromere, while its X chromosome was sub-middle centromere and its Y chromosome was middle centromere with an arm ratio of 1.2, which all were the same as its male parent (Holstein). But Li and Lu (1984) showed that the autosome of yellow cattle–yak was end-centromere which was the same as its parents, and its sex chromosomes were sub-middle centromere and similar to its parents. There were significant differences in the relative length of pairs of chromosomes among parents and yellow cattle–yak, especially in X and Y chromosomes (including arm ratio, p < 0.01).

5. Study on the development of genital organ

The study on the F1, F2, and F3 hybrid cattle by Zhang (2001) showed that the thickness of the white membrane of testes reduced, the blood vessel changed from thin to large, testes became dense and the amount increased, and the cross section changed from dark yellow to faint yellow-white with the increase of hybrid generation. F3 had same testis color and morphology as male yak. On comparing the diameter and the number in each unit, the dissection area of the fine sperm vessel showed that: for the diameter of fine sperm vessel, F3 had an extremely significant difference with F1, F2, and male yak (p < 0.01), but the latter three had no obvious difference (p > 0.05). For the fine sperm vessel number in each viewing field, there was a significant difference between F1 and F2 (p < 0.05), and an extreme significant difference among other groups (p < 0.01). For the area occupied by the substantial tissue of fine sperm vessel in each square centimeter, F1 had no obvious difference with F2 (p > 0.05), but there were extreme significant differences between F3 and male yak, between F3, male yak and F1, F2 (p < 0.01). That means that substantial composition in F3 male cattle's testis obviously increased, but still did not reach normal level. The number of spermatogonia in sperm vessel had no significant difference among F1, F2, and F3 (p < 0.01), but the death rate of spermatogonia differed significantly. That means the death rate of spermatogonia obviously dropped with the increase in generation. There were few deaths occurring in F3, while controlled male yak had no dead spermatogonia. For the number of spermatogonia, F2 and F3 had significant difference with F1 and controlled male yak group, but there were no significant differences between F2 and F3 (p > 0.05) and between F1 and controlled male yak (p > 0.05). It is also found that both hybrids belong to F2, a hybrid male cattle has a part (27.99%) of the spermatogonia showing cell division and could produce sperm cell and sperm, while another male cattle was very poor for sperm production. It indicated that the fertility degree of hybridization varied individually in the same generation. Spermatogonia (39.39%) of F3 appeared by a large amount of cell division, and could produce sperm cell and sperm.

In conclusion, the genital isolation of male sterility of hybridization from yak and ordinary cattle was related to the unhealthy development of fine sperm vessel and the spermatogonia epithelial cell of testis, the death of spermatogonia, the inability of spermatocyte to fulfill meiosis, the unhealthy development of interstitial cell, the low secretion level of gonadotropin and testicular hormone, and the forming of the isolating band between interstitial cell and basement membrane of fine sperm vessel. With the increase of back-cross generation, the development of testis tissue of interspecific hybridization offspring gradually improved and the death rate of spermatogonia gradually reduced. In our experiment, a male hybrid cattle in F3 could produce sperm, for each individual from the same generation there existed differences in sperm development level.

6. Study on the structure of pituitary and endocrinology

Luo (1990) observed the distal part of anterior pituitary of male yak and male cattle–yak by electronic microscope, the results showed that the follicle-stimulating hormone (FSH) cell expanded in cattle–yak, the cell nucleus malformed seriously, the plasma entered into the nucleus, and there was little secretory granule. The variation of FSH cell in male cattle–yak was determined as one of the direct reasons for male sterility. The physiological element for the normal sex behavior of male cattle–yak probably was that the luteinizing hormone (LH) cell of anterior pituitary secreted normal LH content and interstitial cell of testis developed well, while normal LH cell is the physiological element for the normal second sexual character.

The dynamic varieties of LH, T, P4, and 17ß-E2 in 24 hour in blood for male cattle–yak were studied by radio-immunoassay, the content of hormone during day and night was analyzed by t-test, and monofactorial variance analysis of mean level of the above hormones during the day and night was done respectively between cattle–yak and yak, 1/4, 1/2, and 3/4 wild yak. The results showed that the physical element for the normal sexual behavior of male cattle–yak was the normal LH level of the LH cell of anterior pituitary and the normal T secretion level of the interstitial cell of testis.

7. Study on biochemical genetics

The polymorphism of hemoglobin (Hb) of yak, Holstein, hybrid F1 (cattle–yak), and F2 (cattle–yak cross with yellow cattle bull) was studied with polyacrylamide gel electrophoresis (Chang & Wei 2002). The results showed that: (1) the Hb electrophoresis type of Qinghai Holstein displayed polymorphism, while that of Qinghai yak displayed monomorphism; (2) the Hb electrophoresis type of cattle–yak displayed polymorphism with cattle–yak's special band type besides the Hb feature of both parents; (3) the Hb electrophoresis type of F2 was complicated, and it showed four types and also individuals with new Hb type except for Hb feature of its parents; (4) with the increasing of upgrading generation, the Hb electrophoresis type of yak hybrid offspring even became more complicated which should be done further.

The glucose-6-phosphate dehydrogenase (G6PD) of yak, Holstein, hybrid F1, and F2 was detected by polyacrylamide gel vertical plate electrophoresis (PAGE; Chang & Wei 2002) The results indicated that: (1) the G6PD of Holstein and yak all displayed G6PD-A, and no difference existed among individuals, and no significant difference existed between sexes; (2) the enzyme lineage of cattle–yak was basically same with its parental Holstein and yak, all of the individuals displayed a G6PD isoenzyme that was composed by two directly neighbored threads; (3) regardless of whether it was an orthogonal cross (F1 cross with yellow cattle bull) or reciprocal cross (F1 cross with yak bull), the G6PD appeared single or several band, polymorphism existed in individuals judged by the band type.

8. Preliminary study on the RAPD marker of yak, Holstein, and their hybridization

RAPD (random amplified polymorphic DNA) technique was used to analyze the heredity variation between yak and Holstein, yak and cattle–yak (Wei & Zhang 1999). Among the 80 primers that were used, 7 of them expanded segments with clear band and polymorphism, while the other 10 expanded monomorphism for RAPD band type in individuals of yak, cattle–yak, and Holstein. The bands produced by polymorphism primers were between 5 and 8, and the length of segments expanded was between 125 bp and 2322 bp. The results showed that yak had a certain heredity variation and also a certain heredity similarity with Holstein, and the heredity variation between the yak and Holstein was minimal than that of cattle–yak group.

9. Study on the relationship between meiosis and male sterility of yak hybrid

Synaptonemal Complex (SC) is a trilineate structure formed with homologous chromosome at the prophasepachytene of the first meiosis during the maturation process of sexual cell. It has a close relationship with the homologous chromosomal pairing and recombination and the chromosomal agglutination and disjunction during the first meiosis. It has been gradually used in the study of pairing behavior during the meiosis prophase of human chromosome, and many kinds of abnormal pairing have been found.

Under the light microscope, male cattle–yak cells form an SC structure (Zhou et al. 1999). The SC of autosome appeared to be a long and thin monolinear structure with a dark dyed spherical centromere. Many knobs are found in the synaptonemal autosome, which indicated that the exchange between the paired autosome was very possible. The SC has more fractures or even breakages and disintegration. More abnormal phenomena of meiosis and SC appeared in male cattle–yak, which indicated that the normal synapses exchange and segregation of chromosome was disturbed and/or destroyed by the structural differences of two sets of chromosome of male yak–cattle. The SC of sexual chromosome mostly showed linear type or with a few of twisted type. Male F2 yellow cattle hybrid has much more cells to form SC structure than cattle–yak, although without much difference in the basic structure, the shape is more obvious, and the broken and smashed rate of SC also reduced, which indicated the synaptonemal effect of F2 yellow cattle hybrid was better than that of cattle–yak, but with many malformed sperms in observation. More SC can be found in F3 and their shape was almost as same as that of normal cattle, but some fragment, disintegration and unbalanced segregation of chromosome still could be found, the SC of sex chromosome appeared most linear and occasionally twisted.

Spermatocyte, spermatocyte II, spermatoblast, and unhealthy developed sperm were observed by the micro-observation on meiosis cell of cattle–yak testis, and spermatocyte, spermatocyte I, and spermatocyte II were in majority. A part of spermatocyte could finish all processes of meiosis and could produce sperm. That means leptotene, synaptene, pachytene, diplotene, and diakinesis/mid-phase I are successively passed during the prophase of meiosis I, and the metamorphic development process of spermatoblast to the sperm-forming stage is observed. The sperm development of two male hybrids F2 improved obviously than that of male cattle–yak. It is observed that many spermatocyte could finish all processes of meiosis and produce sperm, but most malformed sperm that showed nonterminal cap, slender neck, nonsheath membrane in tail, uncovered tail fiber, and tail looking like two tails branched into two strands from middle piece, main piece, and end piece. SC and mature or non-mature sperm were observed in one F3 cattle. The synopsis, intersection and the segregation of chromosome and the sperm developing process were all close to that of normal cattle, but the function of meiosis and sperm occurring capacity was still inhabited by many malformed sperm.

The gradual development of male yak fertility of hybrid offspring has been proved by many studies, and the gradual development depends on upgrading generations, such as “With the increasing of grading generation, the fertility of hybrid yak recovered,” while the fertility degree of variation among individuals of the same hybrid generation is neglected or denied. That leads to phenomena of resisting the fertility of male hybrid when it appeared in lower generation (F2 and F3). The fertility variation among individuals has been proved by Zong (1985) in the diversity of sterility and the gradual development of the mule's fertility. The study proved that the gradual developing of male fertility of yak hybrid progeny existed not only in different upgrading generations, but also individually in the same generation. Zhao (1998) detected the SC of 11 yellow cattle F2 hybrid, which proved that the variation of sperm occurring level and SC existed among individuals although they all belonged to F2 (Zhao 1998). Among the 11 F2 hybrids, 3 were observed with more SC and sperm, 4 with SC and malformed sperm, and 2 with few of sperm and malformed sperm. Zhang and Duan (1991) collected semen from 2 F3 hybrids and found one with normal sperm while the other with no sperm. Zhao (1998) studied 3 F3 hybrids, and found 2 of them had better meiosis and sperm producing level than the other one. This proved that the fertility recovering of male hybrid existed in upgrading generation.

10. Study on the genes correlating spermatogenesis

Modern molecular biology provides an opportunity to study the genetic mechanism of cattle–yak male sterility in recent years. Some sterility-like genes, which might be candidate genes in male infertility of cattle–yak, have found by some of scholars. Real-time polymerase chain reaction (PCR) analysis indicated that the expression level of genes mRNA in yak testes, which included b-Boule (one of DAZ family gene), Cdc2 and Cdc25A (Cell division cycle), Dmc1 (disrupted meiotic cDNA), SNRPN (small nuclear ribonucleoprotein polypeptide N), SYCP3 (Synaptonemal complex protein 3), and H19 (a non-coding RNA), was significantly higher than that in cattle–yak (Dong et al. 2009; Zhang et al. 2009; Li et al. 2010; Pan et al. 2010; Wang et al. 2012). These results suggest that candidate genes were possibly associated with disturbed spermatogenesis of male cattle–yak, and might contribute to the sterility of F1 male hybrids between cattle and yak (Li et al. 2012).

In the contrary, the lower expression of lactate dehydrogenase C (ldhc), Insulin-like growth factor 2 (IGF2), and Deleted in Azoospermia-Like (DAZL) genes in cattle–yaks' testes than in their parents were also regarded as the reasons of the sterility in F1 (Liu, Li, Zhang et al. 2009; Liu, Li, Pan et al. 2011; Huang et al. 2012). Both DAZL and IGF2 were essential for spermatogenesis. The higher methylation level of the IGF2 and DAZL was irrelevant to the lower expression in cattle–yaks (Liu, Li, Zhang et al. 2009; Liu, Li, Pan et al. 2011). The proportions of the ldhc variants assayed differed significantly among adult yak, yak calf, and cattle–yak; more ldhc transcripts were spliced in immature or sterile testes. These studies showed that ldhc, IGF2, and DAZL play an important role in bovine spermatogenesis and might be involved in cattle–yak male sterility. However, the regulation mechanism of those genes during reproduction is poorly understood. Further work is needed to elucidate the function of those genes in spermatogenesis.

11. Studying outlook about male sterility of cattle–yak

The study on the male sterility mechanism of cattle–yak is always a focus problem for animal husbandry worker. An intensive study on molecular genetics should be done using SC technique, DNA fingerprint, mtDNA, enzyme, and polymorphism technique of blood protein. The variation of heredity structure between yak and ordinary cattle should be confirmed in order to provide the accordance for studying of male sterility mechanism. The male hybrids should not be eliminated too early. Gradual development of fertility of back-crossed male hybrid and looking for fertile male hybrid with special heredity background from the lower generations, paying attention to the physical appearance of the testis development, and checking meiosis and sperm occurrence level by biopsy. Meanwhile, applying subsidiary techniques such as injection of exogenous hormone, feeding, management, and intensive training is among the effective ways in overcoming the male sterility of cattle–yak.

Funding

This research was supported by the Key Projects for Technology Integration and demonstration of Production and Ecological and Life in Northwestern Sichuan [2012BAD13B06] and National Beef Cattle Industrial Technology System [CARS-38].

Additional information

Funding

Funding: This research was supported by the Key Projects for Technology Integration and demonstration of Production and Ecological and Life in Northwestern Sichuan [2012BAD13B06] and National Beef Cattle Industrial Technology System [CARS-38].