Indian Journal of Experimental Biology Vol. 42, November 2004, pp. 1043-1055

Review Article

Fertilization, embryonic development and oviductal environment: Role of estrogen induced oviductal glycoprotein

Purvi Bhatt*, Kaushiki Kadam, Anjana Saxena* & Usha Natraj**

National Institute for Research in Reproductive Health (ICMR) Jehangir Merwanji Street, Parel, Mumbai 400 012, India

Mammalian oviduct is the physiological site for sperm capacitation, gamete fertilization and early embryonic development. The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP) in response to estrogen. The protein has been shown to interact with gametes and early embryo. Several key functions have been postulated particularly its role in pre-implantation events which would have far reaching implications in assisted reproductive technology and in the development of non-hormonal contraceptive vaccine. The intention of this article is to discuss the current status of the protein and analyze how far the postulated function of OGP has been borne out by the available data.

Keywords: Embryonic development, Estrogen, Female reproduction Glycoprotein, Oviductal glycoprotein

Fertilization is a process whereby individual gametes from the female and male unite to create offspring whose genetic makeup is different from both the parents. The oviduct or Fallopian tube (Fr), named after its discoverer Gabriele Fallopious 1 occupies a unique position in reproduction. The oviduct provides an environment that supports the gametes, the process of fertilization, early embryonic development and delivery of a viable embryo to the uterus. It is the first environment to which the embryos are exposed till it enters the progestationally receptive uterus 1•

Oviduct can be broadly subdivided into three sections viz. the infundibulum or fimbria, the ampulla and the isthmus. Fimbriated infundibulum is a trumpet shaped organ comprising the distal portion of the oviduct opening into the peritoneal cavity, positioned to facilitate ov um pick-up from the ruptured follicle and propel it into the ampulla. The ampul la, which is the longest portion of the oviduct, is the site of fertilization, early embryonic development and major biosynthetic activity. The isthmus which extends further from the ampulla ends as a uterotubal junction and functions as a sperm reservoir, regulating sperm transport to the ampulla as well as passage of

**Correspondent author: Tel: 91 -022-4132111/2/6/7, Fax: 91 -022-4139412 Email: dirirr @vsnl.corn; ushan3@rediffmail.com *Present address- New York University School of Medicine. New York . USA

the embryo(s) into the uterus. The outermost serosal iayer consists primarily of ciliated cells and secretory non-ciliated cells. Differentiation and ciliation are induced by estrogen (E2) but is attenuated or blocked by progesterone (P4) in the human2

-4

, baboon5 and cynomolgus macaques6

. The presence of receptors for estrogen and progesterone have been well documented7

'8

. A small population of cells referred to as indifferent cells, lie deep along the base of the epithelium and may be progenitors of the epithelial and stromal elements or function in immunological defence4

.

Fertilization does occur in vitro (IVF) in a simple medium and pregnancies have been achieved which bypass the need of oviduct for these processes, thereby questioning the role of oviduct and/or its secretions. The pregnancy rates have not changed over the years despite improved IVF culture medium that allows development till blastocyst stage, indicating support for the contribution of oviductal secretion. It has been observed that less than 50% of embryos reach the blastocyst stage and of this only 25-35% are capable of establishing viable pregnancy9

.

Even among groups of human embryos capable of reaching the blastocyst stage only few are implanted. Additionally, there are morphological , metabolic and molecular variabilities in the quality of human embryos . Some of the cellular abnormalities that contribute to lower pregnancy rates are lack of cell

1044 INDIAN 1 EX? BIOL, NOVEMBER 2004

mass, high rate of cell death , thinning of zona pellucida and inability to initiate hatching 10

. Further, majority of early embryos cultured in vitro in a chemically defined milieu soon reach the arrest phase of cell cleavage, which occurs at different cell number in different species 11 . These observations suggest that certain factors necessary for embryonic viability and growth are lacking in the culture medium. Indeed, many in vitro co-culture studies with oviductal epithelial cells have shown improved embryo development and pregnancy rates in human and ovine 12·1' eggs fertilized in vitro. Co-culture studies in pigs 14 , cattle 15, and sheep 16 have also shown that oviductal epithelial co-culture (OEC) or conditioned medium (CM) of oviductal tissue culture promoted embryo development in vitro.

From these findings, it can be inferred that oviduct is not a passive tube merely providing physical support to the gametes but is an active organ, which produces factors that facilitate egg-sperm interaction and early embryonic development.

Oviductal fluid composition Oviductal fluid forms the medium wherein the

reproductive processes take place. There are two views expressed regarding protein secretion and accumulation of proteins within the oviduct. First, the oviduct provides a passive source of protein, derived from serum and other fluids as a transudate 17'18. Major components of this transudate being serum protei ns such as albumin , transferrin , immunoglobulins along with enzymes and other prote in molecules, including cytokines, growth factors and transport proteins. Second, the oviduct is also seen as a biologically active, dynamic tissue synthesizing and releasing macromolecules into the lumen throughout the estrous or menstrual cycle. Under normal physiological conditions, it is not feasible to obtain sufficient oviduct tluid for study from human subjects due to ethical considerations. Firstly, an indwelling catheter must remain in the lumen during collection and secondly, surgery in presence of the catheter and any negative pressure applied to withdraw the fluid affects both the volume as well as composition of oviduct fluid. Inspite of all these limitations, the composition of oviductal fluid has been studied and documented in humans and few other species 17-23

. Many proteins have been found to be synthesized in the oviduct including specific protease inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1 24 and plasminogen activator inhibitor (PAI)-1 25 . The evidence concerning

the presence of these proteins has been derived from RT-PCR , Northern blot analysis , in situ hybridization or immunolocalization studies and RIA or ELISA determination of the specific factors in the oviductal fluid or oviductal tissue/cell-conditioned media. Apart from these, many key hormones like Prostaglandins

PGF2a and PGE26, acetycholine, neurotensin and oxytocin27 are also present which control contraction and motility .

During the characterization of ov iductal fluid, investigators have focused attention on those proteins that interact with gametes-either sperm or eggs, since this interaction would have some physio logical relevance. Several methods were used to identify oviduct specific proteins. Use of immunoaffinity technique to remove serum proteins, followed by classical protein fractionation led to the identification of four proteins of molecular weight 44- I 50 kDa, from human oviductal fluid. Interestingly , these proteins could interact with sperm28

. One of the

proteins identified was a-fetoprotein. a known hepatic protein29 . This has been verified in our laboratory using mono-specific antibodies to immunoprecipitate the newly synthesized and released protein in short term human oviductal minces30. Analysis of monkey oviductal protein31 has also revealed the inc reased content of high molecular weight protein during menstrual cycle. Variation in the ov iductal protein pattern on SDS PAGE during estrus cycle in rabbit has demonstrated estrogen and progesterone induced novel proteins32

. Presence of estrous associated 80-90 k:Oa protein has also been noted in sheep·n Identification of newly synthesized proteins in short­term oviductal minces has yielded better insight o n the identification of stage specific proteins. Presence of estrogen regulated high molecular weight glycoprotein has been noted in several species . I d. 34->6 mc u 1ng women · .

One of the most abundant protei ns synthesized within the oviduct are called oviduct specific glycoprotein, OGP. This family of proteins is shown to be estrogen dependent and oviduct-specific . The protein is conserved across the species and is present during the time of ovulation, fertilization and pre­implantation period . Recently, in silico analysis of zona pellucida proteins, ZP2, ZP3 and OGP sequences was carried out to analyze positive selection pressure and sequence conservation. It was noted that these three proteins from the female reproductive tract were the most conserved and

BHATI et al.: ROLE OF ESTROGEN INDUCED OVIDUCTAL GLYCOPROTEIN 1045

positively selected. Among these three, OGP was the most conserved with little sequence diversity as compared to that of ZP2 and ZP337

.

Our research is focused on estrogen induced major oviductal glycoprotein, OGP. The intention of this article is to discuss the current status of the role of this protein and analyze how far the postulated function of OGP has been borne out by the available data.

The postulated functions of OG P- ( i) Association of OGP with egg, zygote and blastocyst would inhibit implantation within the oviduct; (2) Association of OGP with embryo and blastocyst would enhance implantation leading to improved pregnancy outcome; (3) Interaction of OGP with sperm would positively improve sperm function; (4) Potential candidate for the development of a non-hormonal contraceptive; and (5) As an immuno-modulator.

Before we scrutinize in the detail data supporting some of these hypotheses put forth, it is pertinent to examine the characteristics of OGP.

Oviduct specific glycoprotein Oviductal glycoprotein, one of the major family of

glycoproteins identified in a variety of mammalian species is called variously as oviduct secretory glycoprotein, oestrus-associated glycoprotein, oviduct­specific, estrus-associated glycoprotein (EGP), oviduct glycoprotein (s0P92), oviductin, MUC-938

,

glycoprotein GP-215 and oviduct-specific glycoprotein (OGP) all identified collectively as oviduct-specific, estrogen-dependent glycoproteins (OGPs). These are relatively high molecular weight proteins, secreted into the lumen of oviduct ranging from 70 to 350 kDa (mouse, 215 kDa39.4°; hamster, 160-350 kDa4143

; rat,

>330 kDa44; rabbit, 71 kDa45

; pig, 75-85 and 100 kDa46

.47

; sheep, 90-92 kDa33.48; cow, 85-97 kDa49

.51

;

baboon, 100-130 kDa5; and human, 130 kDa52

) . These are exclusively of oviductal origin synthesized and released by oviduct ~ecretory epithelium in all mammals examined. Recently, the endocervix has been demonstrated to be the site of its synthesis53

. The significance of its elaboration needs to be established.

Molecular cloning of mammalian OGP: Evolutionary conservation

In the early 90's the pioneering work of Verhage and his co-workers have contributed enormously in the characterization and functional studies of this class of molecule. Major breakthrough in this area was in the preparation of mono-specific rabbit antibodies to baboon and human OGP in late eighties . With the availability of this antibody hormonal regulation of this protein and association of OGP with gametes could be analyzed. More importantly, molecular cloning of eDNA could be attempted. The cloning of full length human OGP was carried out in 1996. Since then, most of tthe mammalian OGPs have been cloned and the nucleotide sequence based analysis is listed in Table 154

.62

. The open reading frames of eDNA code for a pre protein with 21 amino acid signal sequence and a total protein of 527-721 amino acids. The predicted molecular masses range from 58.5 to 76.5 kDa, which is considerably less than-the calculated mass of reduced denatured glycoprotein. The immunological evidence for evolutionary conservation of OGPs across the species could be inferred by the data presented in Fig I. In Western blot analysis, the antibodies developed against human OGP interacted with mouse and

Table 1--...{::haracteristics of mammalian oviductal glycoproteins

Species Accession mRNA Protein Secret Association Stage & region specificity number (kb) ORF" (kDa) Ion (kDa)

Human U09550 2.4 75.4 130 ZP, PVS Periovulatory throughout oviduct Baboon M59903 2.4 69.3 120 ZP, PVS Periovulatory throughout oviduct Rhesus U872529 2.4 69.3 120 Not known Periovulatory throughout oviduct Bonnet AF484550 >2.0 73 Not known Sperm head and tail Periovulatory

AY341432 Sheep Ul6719 2.2 58.7 90-92 ZP of eggs Blastomeres sperm Present throughout the cycle &

highest at periovulatory Not Isthmus· Pig U43490 2.2 58.5 75-85/100 ZP, PVS Present throughout the cycle &

highest at estrus Not isthmus Cow Dl6639 2.2 59.6 97/90-95 ZP & sperm head Present throughout cycle & highest at

estrus Hamster Ul5048 2.5 69.4 160-350 ZP & sperm head Present throughout cycle, Estrogen

Mouse D32137 2.9 78.8 >215 PVSb(not ZP") dependent Estrogen dependent Not Isthmus

ORF- open reading frameb PVS: perivitelline space< ZP: zona pellucida

1046 INDIAN J EXP BIOL, NOVEMBER 2004

monkey OGP. Recombinant monkey OGP could be immunostained with antibodies to human, baboon, monkey and porcine OGP [unpublished data]. Yeast expressed mouse glycosylated OGP could be immunostained with antibodies to monkey and human OGP. Human, baboon, cow, pig, sheep, and hamster OGPs consist of several molecular weight forms and at least two isoforms, acidic and basic molecules. Bovine OGP consists of seven isoforms63 while pig OGP demonstrates 3 major isoforms and two molecular weight variantsM. A remarkable feature amongst the eDNA sequence of OGP of all the above species is that

A

B

7G 50

2 3

2

D II

2

- 250 kOA ....

,._ 160 kDA •

c

+- 250 kDA

+-160 kDA

3 4

Fig. 1- lmmunological evidence to demonstrate the evolutionary conservation of mammalian OGP by western blot analysis.

[1] The ability of antibodies to monkey and human OGP to immunostain yeast expressed mouse glycosylated OGP. The membrane was probed using rabbit antibodies to human and recombinant monkey OGP. (A)-Blot probed using antisera to monkey OGP; (B)-Blot probed using antisera to human OGP. [Lanel-vector; 2-clone 4; 3- clone 15] and (C)-B lot probed using DIG glycan detection kit. [II] depicts the abi lity of antibodies to human, baboon, monkey and porcine OGP to immunostain bacterially expressed monkey OGP. [D- recombinant Bonnet monkey OGP Western blotted and probed with AJ_S to human, baboon, bonnet and porcine OGP].

they exhibit significant similarity (86-90%) and high degree of homology (77-84% identity) in both nucleotide and amino acid sequences in the amino terminal end when compared to carboxyl terminal end. Major sequence differences are localized in the carboxyl terminal region52

, which coJTesponds to exon 11 of the gene.

The organization and structure of genomic sequences have been described for human65

, mouse66

and hamster67 OGPs. The sequence is contained in chromosome I. The coding sequence is contained in II exons with the signal sequence localized in exons 2 and 3 and the mature peptide found in exons 3-1 I. A TAT AA box has been identified in human 5' flanking sequence while the mouse and hamster lacked typical TATA, CAAT and CG boxes. An imperfect estrogen response element has been detected in hamsters and mice and ten half palindromic ERE have been located in the putative promoter in mice.

Structural similarity of OGP with glycosyl hydro lases All the OGP's sequenced to date, demonstrate

simila.r~ty to a growi ng group of mammalian gene products, which include human cartilage· glycoprotein 3968 and human macrophage chitotriosidase69

, and a group of enzymes (chitinases) classified as Family 18 glycosyl hydrolases with no detectable enzymatic activity70

. It has been hypothesized that the chitinase­like active site may retain its sugar binding property.

Post-translational modification of OGP The presence of tandem repeats typical of mucin

family could be seen in the deduced amino acid sequence of OGP. However, there exists a species variation. Thus, hamster7

I.72 has six repeats of 15

amino acids and mouse 21 repeat sequences of seven amino acids each64

. In the case of human, baboon and rhesus monkey four tandem repeat sequences could be seen. On the other hand, cattle, sheep and pig show in complete or no tandem repeat. The carboxy terminal region has potential N (1-7) and/0-linked glycosylation sites ranging from 10 to 20 except in case of mouse OGP which has 67 predicted sites. If glycosylation occurs at all these sites one could expect the secreted protein to have large molecular size. This could account for the observed differences in molecular size of mammalian OGPs even though the ORFs were not very different.

Carbohydrates commonly found include terminal N-acetyl neuraminic acid (sialic acid) in a (2,3) and a (2,6) linkages, galactose, N-acetylgalactosamine, a-

BHA TI eta/.: ROLE OF ESTROGEN INDUCED OVIDUCTAL GLYCOPROTEIN 1047

fucose, galactosyl-~( 1.3)-N-acetylgalactosamine, N­acetyl g lucosamine and mannose64

·73

. Metabolic labelling studi es have shown that in rabbit45

, pig64 and hamster71

'72 OGPs are modified by the addition of

inorganic sulphate. Phosphorylation of OGP has also been shown in pig 74

. The heterogenous addition of sialic aci d other sugar residues, sulphation and phosphorylation cou ld res ult in variable isoforms of OGP within a species. The biological significance of these iso forms needs to be evaluated .

Hormonal elaboration of OGP With avail abil ity of mono-specific antibod ies and

eDNA sequence data of OGP it was possible to examine the hormonal regulation of OGP at the mRNA and protein level. Immunolocalization to analyze the stab ly expressed immunoreactive protein in a particular cell type and in situ hybridization as measure of RN A expression in a particular cell type was studied. It was demonstrated that OG Ps are products of non-c ili ated oviductal epithelium. Regional di fferences in the expression of OGPs appeared to be a dominant feature in some species. In primates, OGP is expressed in the in fundibulum, ampulla and isthmus-": In golden hamsters75

, OGP expression is seen in ampull a and isthmus whereas in sheep and pi g64

, it is present in ampulla and infundibulum76

. The regul ation of expression appeared to di ffer among species since the protein(s) are continuously present in the oviductal epithelium at all stages of the cycle and during pregnancy of mature golden hamsters77 and mice78

. Suppress ion subtractive hybridi zation and northern hybridization have also identified OGP as one of the differentially expressed genes in mouse pre-implantation period79

. E2

admini stration to pre pubertal hamster induces OG P expression80

·8 1

. Similar situation prevalent in pre pubertal mice is shown in Fig. 2. The expression of mouse OGP was ascertained by RT PCR79

. In case of primates production is turned on by es tradiol and off by progesterone3

1. 56

. The levels of OGP expression in regularly cycling bonnet monkey shown in Fig. 3, the support the same57

. Levels of OGP measured have been found to be temporally associated with elevated levels of E2 during the estrous cycle and early pregnancy in the pig64

, cow82·83 and sheep3 1

•84

.

Ovariectomy and hormone replacement with E2

induces expression of OGP mRNA and protein, whereas P antagonizes the effect of E2 in , baboon85

,

rhesus monke/ 6, pig86 and sheep35

·76

. Although , the hypothesis that induction and upregulation of OGP

ex press ion is Erdependent that is supported by these studies, several laboratories have demonstrated that LH receptors are present and functional in human87

,

OGP

ER

PR L19

1&0

160

140

12 0

10 0

80

60

~

20·

0

B O oGP -

-

-

n co ntrol

24hrs 48hrs 72hrs

Time (hr) following estradiol treatment.

@ ER

0 PR

., I

control 24hrs 48hrs 72hrs

Time {hr) following estradiol administration

c

380bp

325bp

350 bp 200bp

Fig 2-Hormonal regulation of OGP. Expression of OGP, ER and PR in the oviduct after estrogen treatment at 24, 48 and 72 hr. [Panel A] Level s of OGP, ERa, PR and L-19 analyzed in the oviduct by RT-PCR from 22 Day immature mice injected wi th 10 11 g estradiol and sacrificed at 24, 48 and 72 hr post treatment. ; C­control untreated, [Panel B) Plot of relative intensities of OGP, [Panel C] ER<><, PR to L- 19 [house keeping gene]. The agarose electropherogramme was scanned [UMAX Powerlook 1100 scanner] and analyzed [Image SXM software] . The altered expression of the gene: ER<><, PR and OGP was normalized to the expression of L/9, the house keeping gene. OD of the gene /L/9 x 100.

1048 INDIAN J EXP BlOL, N_9VEMBER 2004

E2 1evels (pg/ml)

OGP expression levels by RT-PCR

<75 >150 >200 <75

Fig. 3--DGP expression in bonnet monkey by RT-PCR [Lanes 1-4: Days 5, early proliferative; days 9, late proliferative; days 12, periovulatory and days 16, mid-luteal].

pig88 and bovine89 oviduct. An increase on OGP mRNA half life in response to hCG89

·90 on bovine

oviductal epithelium suggested a direct effect of this hormone on modulating OGP elaboration .

OGP association with eggs In order to thoroughly understand the function of

OGP in fertilization and early embryonic development, the availability of well-characterized OGP preparation and a good animal model to study in vivo and a good in vitro system are an essential pre­requisite. The ideal preparation would be a protein purified from a natural source obtained from animal at specific stage. This is required because it is known that the protein is hormonally controlled and elaborated in stage specific manner and subjected to post-translational modification. Unfortunately, it is difficult to meet these requirements. Human hydrosalphinx fluid used as a source for obtaining enriched preparation of OGP is far from ideal source material as it is from pathological condition. Our experience in the use of hydrosalphinx as a biological source has been disappointing. For one, not all women accumulate OGP in terms of its concentration to the same extent. There is also variation on the molecular size of the immunoreactive protein as assessed by Western blot which can vary from 110 to 68 kDa. Other enriched preparations available are porcine, bovine and hamster. In our lab, we have utilized recombinant approach to prepare glycosylated and non-glycosylated OGP. We have succeeded in preparing both glycosylated and non-glycosylated mouse and monkey OGP respectively (Fig. 1). Even­though, it circumvents the need to obtain large amount of fluid from natural source, it also suffers from excessive glycoyslation as it occurs in yeast expression system etc. The major advantage is that well-characterized homogeneous preparation could be prepared. Many questions related to the issue of OGP association such as (a) Is it through protein backbone or is it through carbohydrate residues? (b) Is there a separate and distinct binding domain on OGP that

interacts with sperm and zona/egg? could be answered.

The hypothesis that OGP interacts with embryo was verified using indirect method of immunofluorescent or immunogold which examines the presence of immunoreactive protein. The association of OGP with the zona pellucida and perivitelline space of oviductal oocytes and embryos in baboon92

, cow93·94

, sheep 48·94

,

pig95 and hamster96·97 has been amply demonstrated. In

mouse, the binding is limited to the perivitelline space40

. Distribution of OGP in the zona pellucida and its association with the embryos until day 7 of pregnancy in cattle92 and until pig embryos hatch has been recorded95 and this raises an intriguing question whether OGPs associated with embryo undergo modification during development. Experiments could be designed with the use of the lectins with different sugar specificity. Recently, Yong et a /. 98 demonstrated the inhibition of development of 2 cell embryos in presence of antibodie<> to rabbit OGP indicating the role of OGP in the release of early embryonic development block.

Association of OGP with sperm Of the million sperm ejaculated in natural mating,

only thousands reach the isthmus of the oviduct and these are held back in reservoir and only few reach the ampulla, (the site of fertilization) at the time of fertilization. A direct interaction of sperm with the apical plasma membrane that line the epithelial cells of isthmus has been elegantly demonstrated99

: It has been suggested that oviductal. reservoir 100 serves these functions- (1), prevention of polyspermy by controlling sperm transport to the ampulla; (2) , maintenance of sperm fertilizability; and (3) , modulation of capacitation. Since capacitated state reduces the life span of spermatozoa, binding of sperm and their release from the oviductal epithelium is a crucial step in the process of capacitation in vivo. Capacitation is also controlled by Ca2

+ ions. The 'key player in sperm oviduct interaction is carbohydrate moieties. The sugars involved are species specific. The involvement of fetuin in the process of sperm capacitation has been elegantly demonstrated in hamster. In response to changing environment of oviduct, spermatozoa may shed their protein coat creating a new surface which could allow spermatozoa to be released from oviduct. Following detachment from the epithelial cells, the sperm show hyperactive motility and undergo rapid acrosome reaction 100

-102 acquire hyper activated

motility necessary for moving to the site of

BHATT eta/.: ROLE OF ESTROGEN INDUCED OVIDUCTAL GLYCOPROTEIN 1049

fertilization. In support of this is the data with co­culture studies with bovine sperm. The appearance of some newer proteins analyzed ··by 20-electrophoresis clearly suggested changes in secretory activity of the epithelial cells upon sperm binding 103

• The chromatin structure of sperm was found to be better preserved in vitro when co-cultured in the presence of oviductal epithelial cells 104

• In Australian marsupials, sperm co­cultured with oviductal epithelial cell (OEC), monolayers demonstrated thumbtack orientation and high motility, a characteristic considered as an indicator of sperm capacitation 105 again indicating the presence of oviduct component required for capacitation-like changes. There are reports in ram 107

and bovine108 where spermatozoa undergo acrosome modifications or acrosome reaction during co-culture with oviduct epithelial cell monolayers.

From the foregoing studies, it is clear that there are proteins rich in carbohydrate residues or carbohydrates secreted by the oviduct which interact directly with the sperm. This interaction could regulate the physiological status of spermatozoa before ovulation by binding to the plasma membrane overlying the sperm head, and acting as acrosome stabilizing factors and thereby preventing sperm agglutination. Also this association could act as a protective barrier in dilute suspensions of sperm from the leaching influence of oviduct fluid and have a role in hyper activation. Interestingly, OGP has sequence homology to zonadhesin which contains a mucin-like domain located next to the N-terminal region. Zonadhesin gene is restricted to the testis . The eDNA encodes a precursor protein which may be processed during epididymal transit and/or capacitation to generate the mature protein by removing the N­terrninal and mucin-like domains. Zonadhesin has been shown to bind in a species specific manner to the homologous zona pellucida. Hence, the significance of this homology needs to be examined.

If OGP is indeed the oviductal protein that interacts with sperm, it should be possible to show its interaction with sperm as well as demonstrate its effect on sperm function in vitro. This hypothesis has been verified by immunofluorescent studies using purified hamster OGP. Presence of OGP binding sites on the acrosomal region of spermatozoa before and after capacitation 109 was demonstrated. Furthermore, OGP binding to bovine sperm head and mid tail regions differed from that observed in hamster spermatozoa109

• In contrast, no OGP binding with human spermatozoa110 could be demonstrated. On the

other hand, we have demonstrated localization of OGP binding sites onto bonnet monkey sperm head and tail region 111 (Fig. 4). Removal of sialic acid and N-linked glycosylation significantly reduced the ability of bovine OGP to maintain sperm viability highlighting the significance of carbohydrate part of OGPn. The identity of OGP binding sites on sperm has been studied in our lab by screening human testis and epididymal library using recombinant monkey OGP as a bite. A positive clone was identified. Nucleotide sequence of this showed 98% homology to vascular adhesion molecule 1 (V AP-1 ), a gene product which also has copper containing monoamine oxidase activity (Kadam et al. unpublished work.) The possible function of this will be discussed under the section of immunomodulation.

OGP, a candidate for immunocontraception? Ability of OGP to interact with gametes and

fertilized eggs could form the basis for developing non­hormonal imrnunocontraceptive vaccine. [mmunocontraceptive vaccines based on zona pellucida proteins have been attempted and a vaccine based on this has been successfully used for animals 11 2

-

115. The major drawback with this approach is that it leads to complete ovarian failure. Any vaccine which spares ovary would therefore have an advantage and OGP scores over other egg proteins. The vaccine based on this will be acceptable even if its use would be restricted to animals.

As a first step towards such development, effect of preformed antibodies to OGP on sperm egg interaction was carried out. Antibodies to peptide corresponding to the highly conserved region of OGP could inhibit sperm egg interaction in vitro 116

•

Recently, Yong et al. demonstrated the inhibition of development of 2 cell embryos in presence of antibodies to rabbit OGP indicating the role of OGP in the release of early embryonic · development

Fig. 4-Washed monkey sperm were incubated with OGP and OGP binding sites were immunolocalized rabbit anti bonnet monkey rOGP. OGP binding sites could be seen on sperm head and tail in panel (b) and panel (a) represents sperm incubated wi th preimmune sera. Magnification XIOO. [Reproduced from Natraj et at, Bioi Reprod, 67 (2002) 1897].

1050 INDIAN J EXP BIOL, NOVEMBER 2004

block98. We have attempted to extend this study by

conducting both active and passive immunization studies using recombinant OGP as an antigen and mice as an animal model. When antisera to OGP was injected in pro-estrous female mice and mated, no significant difference in implantation site or litter size was seen. We also did not see an effect on the pregnancy outcome when actively immunized female mice were mated. This could be due to inadequate antibody concentration in the oviduct. This was borne out when the antibody titer was measured in the oviduct. It was noted that, even though circulating antibody levels were high, the antibody levels in the oviduct were low [Ill 00111

) to that of the circulating levels. From these preliminary results , it can be argued that unless adequate amount of antibody in oviduct is maintained fertilization would not be prevented and vaccine based on this wou ld not be possible.

Recently, mice null for OGP gene has been created and the physiological function of this gene was evaluated in vitro and in vivo. Data indicated that the fertility of OGP was with in the normal limits 117

• In mice, it appears that OGP is not essential for the process of fertilization. Similar results have been recorded with another gene protein that was thought to be critical for fertilization namely, pro acrosin 11 8

However it should be emphasized that experiments with knockout or null models of gene products identified as candidates for egg binding have failed to provide convincing evidence for any of these proteins. For example, mice null for ~ I ,4- galactosyltransferase (GalTase) have markedly reduced fertility but fertility is not completely elim inated 119

• The process of fertilization is a critical step in the conservation of species . To ensure the continuity of the species, there may be several gene products intimately associated with this process leading to the creation of redundant systems. Therefore, it is very likely that OGP may be part of such a system or mouse may not be an ideal system to assess the role of OGP.·

OGP as an immunomodulatory molecule OGP belongs to family of mucin, MUC 9, and is

thought to encompass features of that family of proteins. Although proven functions for the different mucins are largely unknown, potential functions do suggest that these mucins due to their large extended structures limit accessibility to other cell surface components, thereby exhibiting anti-adhesive characteristics and providing an effective barrier

preventing immune cells from attacking. This probably explains two of hypothetical functions assigned to OGP during fertilization--(a) preventing ectopic pregnancy by covering the zygote by its assocJatiOn, thus acting as an anti-adhesive glycoprotein; and (b) acting as an immunosuppressive factor inhi biting rejection of the embryo by the mother38

. Paradoxically, mucins also promote cell adhesion and therefore, could be potential mediators of sperp1-egg interaction.

It is well-known that majority of sperm are eliminated from the female reproductive tract sooner or later. Only a minute fraction of sperm migrate successfully to the si te of fertilization, ampullar region of the oviduct. In species, in which semen is deposited in the vagi na at coitus, the sperm must negotiate the highly folded mucus filled cervix before entering the uterus. During the periovulatory period the mucus serves to--(1) protect sperm from the hostile environment and phagocytosi by vaginal leucocytes; (2) prevent seminal plasma into uterus; (3) exclude morphologically abnormal sperm; and ( 4) retain and conserve sperm for later migration to the oviduct. Thus, there are two sites in which sperm interact and bind to the epithelial cells namely cervix and oviductal isthmus 120

• Elaboration of OGP by endocerv ix has been demonstrated in rabbit. It is very likely that this could be true with other species as well. V AP-I, a monamine oxidase, has been identified as OGP binding partner in our lab. Expression of V AP-1 in vagina and cervix is also known 121

'122

• One of the functions of V AP-t is leukocyte trafficking resulting in enhanced leukocyte infiltration leading to phagocytosis and removal of dead/damaged cells. It is tempting to speculate that OGP associated with V AP-1 protects the sperm from phagocytosis leading to the preservation of high quality sperm. Evidence, in favour of this speculation is that isthmus region of oviduct elaborate OGP and OGP binding sites are located on sperm. Evidently, more experimental data is required to unequivocally support this contention.

Immunosuppressive role of OGP could be demonstrated easily using peripheral leukocyte with the commercially available kits [and thi s has not been carried out at present] .

Functions of OGP: Assisted reproductive technology The hypothesis that addition of OGP would

improve sperm and embryo quality was assessed in vitro by pretreatment (of sperm) or inclusion of

BHATI et al.: ROLE OF ESTROGEN INDUCED OVIDUCTAL GLYCOPROTEIN 1051

purified or enriched preparation of OGP on sperm functi on, in vitro fertili zation and embryonic development. One fact that emerged from these limited studies was that presence of OGP is required during fertilization for embryotrophic effects to be seen.

In vitro studies in the bovine system, where spermatozoa were incubated with semi purified or enriched OGP showed significant increase in the capacitation rate and fertilizabi lity of spermatozoa 123

•

In another study, using bovi ne sperm increased motility and viab il ity was seen when OGP was added in a dose-dependent manncr 124

. However in the pig 12s 126 . .f. d . I system · , a stgm tcant ecrease 111 po yspermy was

observed at lower concentration wh ile reduced sperm binding and penetration at increased concentrations (~50 ).lg/ml) was seen .

Increased binding and penetration rates were observed in the human and hamster homologous systems 110

'127

. In heterologous systems using human gametes and baboon OGP, despite the high homology between human and baboon OGP, decreased binding and penetration rates were observed suggesting the importance of a homologous system 110

•

In the ovine homologous system, incubation with enriched OGP during fertilization showed significant increase in cleavage rate and number of blastocysts 128

•

Similarly during pig IVF, eventhough, the addition of OGP at fertili zati on showed no effect on the cleavage rate but increased the number of blastocysts 125

•129

.

Embryotrophic effects of OGP were observed in the bovine system when in vivo fertili zed zygotes were treated with OGP in that a significant increase in incorporation of radio-labelled-amino acids into protein at the four-cell stage 130

. Thus, these accumulated data do suggest the invol vement of OGP in fertilization and embryonic development.

From the present studies, it is clear that even though experiments ai med at answering many of the postulated functions have been performed, many questions remain unresollved. The improved embryo quality in presence of OGP in vitro could form the basis of improving IVF media essential in ART. The next question that needs to be addressed would be on the implantation and improved pregnancy rate of such embryos. It is likely this question at least in the farm animals will be answered soon. With the advances in the field of recombinant proteins and methodology for protein purification, it is likely that recombinant OGP could be freely available. With the availability of this,

information on the role of OGP in improving the JVF media in ART will be resolved.

These data wi ll strengthen the posttton of this hi ghly conserved OGP in the area of fertilization and early embryonic development.

Acknowledgement Thank are due to CSIR , New Delhi for awarding

SRF to the authors (PB and KK).

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